Pde inhibitors in immunotherapy

ABSTRACT

The invention features methods and compositions featuring a PDE5 inhibitor for treating or preventing immunological-mediated disease in a subject.

REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application Ser. No.60/642,029, filed on Jan. 7, 2005, the contents of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

Host immunity to cancers has been extensively documented both in animalmodels and humans (1). In fact, there is strong evidence that the immunesurveillance plays a critical role in limiting tumor outgrowth in theearly stages of tumorigenesis (2, 3). However, the ability to primetumor-specific T-cells and sustain an immune response that imparts ameasurable clinical benefit, is limited in the setting of an establishedtumor burden (4, 5). Taken together, these findings suggest numerousrequirements for effective immunotherapy. Tumor-specific T cells mustnot only possess a sizeable precursor frequency and reach sufficientnumbers following activation, but they must also be able to traffic tothe tumor site and effectively kill their targets in situ.

Growing tumors are able to modify their microenvironment and render itmore immunosuppressive. Such intratumoral changes include altering thecytokine milieu, changing the extracellular matrix, and recruitingimmune cells with a suppressive function. In mice, the CD11b⁺/Gr1⁺ MSCsrepresent one population of cells within the tumor microenvironmentresponsible for the immunosuppression accompanying tumor growth (6, 7).Their elimination in tumor-bearing hosts restores CD8⁺ T cellresponsiveness (8, 9). This observation points to a reversible processand supports the hypothesis that strategies aimed at the pharmacologicinhibition of these pathways can be effective in restoring immuneresponsiveness. L-Arginine metabolism is a key pathway used by MSCs toblunt the anti-tumor response both in mice and humans ((10, 11) andSerafini, Noonan unpublished data). Arg1 and NOS2, the main enzymes thatcatabolize L-arginine, can, in fact, work either alone orsynergistically in restrain T-cells response (12). Through anunderstanding of these critical suppressive pathways, it is possible todetermine whether selective immunopharmacologic targeting can augmentanti-tumor immunity. Nitroaspirin derivatives were recently shown todown-regulate NOS2 expression in tumor associated MSCs and to abrogateMSC-mediated immune-suppression in vivo (13) but the mechanisms of theseeffects were not defined. While the transcriptional andposttranscriptional mechanisms regulating NOS2 expression have beenextensively studied, little is known about the pathways regulatingArginase expression.

Agents increasing intracellular cGMP levels can induce either positiveor negative effects on NOS2 in a cell dependent manner (14). Inmacrophages, for example, cGMP analogues inhibit NOS2 expression (15).Phosphodiesterase-5 (PDE5) inhibitors such as (sildenafil (Viagra®),vardenafil (Levitra®), tadalafil (Cialis®)) increase intracellularconcentrations of cGMP with therapeutic implications that include thetreatment of erectile dysfunction, (16) pulmonary hypertension (17) andcardiac hypertrophy (18). The results delineated herein relate to newmechanisms and functions involving PDE inhibitors, thus providing newtherapeutic compositions and methods for treating or preventing diseaseand disease symptoms.

SUMMARY OF THE INVENTION

The invention features methods and compositions for the treatment andprevention of disease or disease symptoms. This invention is based onthe discovery that PDE5 plays an important role in immune regulation.

In one aspect, the invention generally features a method of treating orpreventing disease, disease symptoms, or disease progression in asubject (e.g., a human patient). The method comprises administering tothe subject an effective amount of a PDE5 inhibitor.

In another aspect, the invention provides a method of treating,preventing, reducing, or reversing cancer in a subject (e.g., a humanpatient), the method comprising administering to the subject aneffective amount of a PDE5 inhibitor, where the administration of theinhibitor treats, prevents, reduces or reverses cancer.

In another aspect, the invention provides a method for treating,preventing, reducing, or reversing disease in a subject having or havinga propensity to develop the disease, the method comprising administeringto the subject an effective amount of a PDE5 inhibitor, where theinhibitor treats, prevents, reduces or reverses the disease.

In another aspect, the invention provides a composition for thetreatment of a condition selected from the group consisting of cancer(e.g., multiple myeloma, melanoma, breast, stomach, head and neck,ovarian, colon, prostate, cervical cancer), chronic infection, orhematopoietic reconstitution following chemotherapy, the compositioncomprising a PDE5 inhibitor in a pharmaceutically acceptable excipient,where administration of the composition to a subject results intreatment of the cancer, chronic infection, or hematopoieticreconstitution following chemotherapy.

In another aspect, the invention provides a composition for thetreatment of disease (e.g., any disease delineated herein), thecomposition comprising at least 0.1-200 mg of a PDE5 inhibitor in apharmaceutically acceptable excipient.

In various embodiments of the above aspects, the composition comprisesat least 10, 20, 100, or 150 mg of a PDE5 inhibitor. In yet otherembodiments of the above aspects, the composition provides for thesustained release of the PDE5 inhibitor In still other embodiments, thecomposition provides for release of the PDE5 inhibitor over at least4-8, 8-12, or 12-24 hours. In yet other embodiments of the aboveaspects, the composition consists essentially of a PDE5 inhibitor.

In another aspect, the invention provides pharmaceutical pack comprisinga composition comprising at least 5 mg of a PDE5 inhibitor in apharmaceutically acceptable excipient, where the pharmaceutical pack islabeled for use in the treatment or prevention of disease (e.g., anydisease delineated herein).

In a related aspect, the invention provides pharmaceutical packcomprising a composition comprising at least 5 mg of a PDE5 inhibitor ina pharmaceutically acceptable excipient, where the pharmaceutical packis labeled for use in the treatment or prevention of disease (e.g., anydisease delineated herein).

In various embodiments of the previous aspects, the pack comprises atleast 10 mg, 20 mg, or 100 mg of a PDE5 inhibitor. In other embodiments,the PDE5 inhibitor is provided in a sustained release formulation. Inother embodiments, the composition consists essentially of a PDE5inhibitor. In other embodiments, further comprising written instructionsfor administering the composition to a subject for the treatment orprevention of disease (e.g., any disease delineated herein).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: In vivo PDE5 inhibition downregulates NOS2 in tumor-associatedMSCs. A cohort of mice were challenged with 0.5×10⁶ C26GM cells andtreated (black bars) with sildenafil (20 mg/kg/day) or left untreated(gray bars). After nine days the mice were sacrificed, single cellsuspensions were obtained from the tumors through collagenase treatment,and the tumor-associated CD11b⁺cells were magnetically purified asdescribed in the Material and Methods. A) Intracellular concentration ofcGMP was measured on the lysate of 10⁶ CD11b⁺ cells through acompetitive Enzyme ImmunoAssay (EIA) kit. B) Western blot analysis wasperformed to detect NOS2, Arg-1 and β-actin expression on 0.5×10⁶magnetically purified tumor-associated CD11b⁺ cells. C) NO productionwas evaluated as the concentration of NO₃—NO₂ in the supernatant of 10⁶purified CD11b⁺ cells cultured for 24 h in DMEM. Arginase activity wasdetermined on cell lysates and normalized for the number of cells. D)Purified tumor-derived CD11b⁺ cells were labeled with anti CD11b-APC andanti-IL4Rα-PE. The histogram is gated on CD11b⁺ live cells purified fromeither the spleen of tumor free (no tumor) mice, untreated C26GMtumor-bearing mice (no treatment) or sildenafil-treated tumor-bearingmice (Sildenafil). Data are the average+/−SD of IL4Rα⁺ cells from twoindependent experiments.

FIG. 2: PDE5 inhibition reverts MSC suppressive pathways. Splenic CD11b⁺cells were magnetically purified from BALB/c mice challenged 9 daysbefore with C26GM, and added to CFSE labeled splenocytes containingeither naïve HA-specific CD8⁺ CL4 cells (A) or naïve HA specific CD4⁺6.5 cells (B). The cultures were stimulated for 4 days with the relevantpeptide in the presence or in the absence of sildenafil (50 μg/ml). Theproliferation was evaluated as CFSE dilution by cytoflorimetricanalysis. C) Splenic CD11b⁺ cells were magnetically purified fromC57Bl/6 NOS^(+/+) or from C57Bl/6 NOS^(−/−) challenged with the melanomaB16GM 15 days before and added to CFSE labeled splenocytes containingnaïve OVA specific CD4⁺ T cells. The cultures were stimulated for 4 dayswith the relevant peptide in the presence or in the absence ofsildenafil (50 μg/ml). The proliferation was evaluated as CFSE dilutionby flow cytometry. Data derived from one of two independent experimentswith similar results.

FIG. 3: PDE5 inhibition alone imparts a measurable immune-systemmediated antitumor effect. Balb/c (A) or BALB/c Rag^(−/−) mice (B) werechallenged s.c. with 0.5×10⁶ cells of the indicated tumor. Sildenafil(20 mg/kg/day) was added to the drinking water or given i.p. daily whereindicated. Tadalafil (2 mg/kg/day) was given ip. Tumor size is indicatedas the product of the two main perpendicular diameters measured with acaliper. (C) GR-1⁺ cells were depleted where indicated by i.p. injectionof 200 μg of anti-GR-1 depleting antibody (clone RB6-8C5.3) on day 0, 3,6 after tumor challenge. Best fit of the data was obtained by fourparameter sigmoid curves. Paired T test P value (P^(T)) or one way AnovapValue (P^(A)) are reported.

FIG. 4: Adoptive cell transfer (ACT) efficacy is improved by sildenafiltreatment. BALB/c mice were challenged on day 0 with 0.5×10⁶ C26GM cellss.c. and were either given sildenafil (20 mg/kg/day) in their drinkingwater or left untreated. Where indicated, the mice received 20×10⁶splenocytes from mice vaccinated 7 days before with 10⁶ γ-irradiatedC26GM. Tumor size is indicated as the product of the two mainperpendicular diameters measured with a caliper. One way Anova P value(P^(A)) is reported, The paired T-test (P value=P^(T)) was used tocompare sildenafil vs sildenafil+ACT groups.

FIG. 5: PDE5 inhibition improves the infiltration and activation oftumor specific CD8⁺ T cells A) BALB/c mice were challenged s.c. with0.5×10⁶ CT26WT cells and given sildenafil (20 mg/kg/day) in the drinkingwater or not. The mice were sacrificed 15 days later. The tumors weresurgically removed, fixed with 10% neutral buffered formalin, andstained with hematoxylin-eosin. B) BALB/c mice were challenged with0.5×10⁶ C26GM cells s.c. Half of these mice were treated with sildenafilstarting on day 0. Where indicated, adoptive transfer was performedutilizing 20×10⁶ splenocytes from H2^(d) pIL-2/GFP mice vaccinated s.c.one week earlier with γ-irradiated C26GM. After 9 days the tumors weresurgically removed, treated with collagenase, labeled with anti-CD4 andanti-CD8 antibodies and analyzed by flow cytometry. The percentage ofCD8⁺ T cells was plotted against the tumor size at the time of tumorharvest. Sigma plot was used to fit a 3 parameters exponential decaycurve (y=36.13+92×e^(−5.93x)). Pearson bivariate correlation:P=0.0000002. Data derived from 3 independent experiments. Tumor singlecells suspension were also labeled with anti-CD25 or anti-CD69antibodies (C). Data are expressed as percentage of positive cells gatedon the CD8⁺ population. D) Since the splenocytes used for the ACT werederived from pIL-2/GFP transgenic mice, IL-2 production in theC26GM-vaccinated T cells is reported as percent of GFP positive, CD8⁺ Tcells. Data are derived from two independent experiments. Paired T testP value is reported. E) BALB/c mice were challenged with 0.5×10⁶ C26GMcells on day 0. Where indicated the mice were treated with eithersildenafil, the anti-CD8⁺ depleting antibody (clone 2.43) on day 0, 2,4, and 6, or both treatments. One way Anova P value is reported. Data isreported from one of two similar experiments.

FIG. 6: The impaired lymphocyte proliferation in cancer patients isrestored by PDE5 inhibition. Ficoll-purified PBLs from healthy donors,head and neck cancer patients or multiple myeloma patients werestimulated with anti-CD3 and anti-CD28 antibody-coated beads at a 3:1bead to T cell ratio in the presence or absence of sildenafil (50μg/ml). CD4⁺ and CD8⁺ T cell expansion was measured by flow cytometry 5days later. Data are reported as fold change calculated as: (number ofpositive cells in the stimulated culture)/(number of positive cells inthe un-stimulated control).

FIG. 7: Sildenafil down-regulates IL4Rα expression on purified MSCs.CD11b+ cells were magnetically purified and cultured in media with orwithout Sildenafil (50 μg/ml). Where indicated, INF-γ (25 ng/ml) wasadded on day 2. The cells were harvested at the indicated time, labeledwith anti-CD11b and anti IL4Rα antibodies. The percentage of IL-4Rα⁺cells was determined by flow cytometry collecting 10000 CD11b⁺ cells.7AAD and annexin V were used to exclude dead cells. Results areexpressed as the average of triplicate wells +/− the standard deviation.

FIG. 8: PDE-5 inhibitors reduce tumor growth. Balb/c mice werechallenged s.c. with 0.5×10⁶ C26GM cells. Sildenafil (20 mg/kg/day),tadalafil (2 mg/kg/day) or PBS were given i.p daily. The tumors weresurgically removed and photographed on day 9.

DETAILED DESCRIPTION OF THE INVENTION Definitions

By “PDE5 inhibitor” is meant a compound that inhibits cGMP hydrolysis byphosphodiesterase-5. PDE5 inhibitors preferably reduce PDE5 enzymaticactivity by at least 5% (e.g., 10%, 15%, 20%, 30%, 50%, 60%, 75%, 85%,90% or 95%). Methods for assaying the activity of a PDE5 inhibitor areknown in the art and are described herein.

By “treat” is meant decrease, suppress, attenuate, diminish, arrest, orstabilize the development or progression of a disease.

By “disease” is meant any condition or disorder that damages orinterferes with the normal function of a cell, tissue, or organ.

By “modulation” or “modulating” is meant any alteration (e.g., increaseor decrease) in a biological function or activity.

By “reduce” or “increase” is meant alter negatively or positively,respectively, by at least 5%. An alteration may be by 5%, 10%, 25%, 30%,50%, 75%, or even by 100%.

By “subject” is meant a mammal, such as a human patient or an animal(e.g., a rodent, bovine, equine, porcine, ovine, canine, feline, orother domestic mammal).

An “effective amount” is an amount sufficient to effect a beneficial ordesired clinical result.

The term “hydrate” means a compound of the present invention or a saltthereof, which further includes a stoichiometric or non-stoichiometricamount of water bound by non-covalent intermolecular forces.

A “Marker” is any compound (e.g., molecule, protein, nucleic acid) orportion thereof (e.g., atom, functional group) or physicalcharacteristic (e.g., radioactivity, binding, energy emission) that ismeasurable and whose presence, absence, or quantification is useful toprovide an indication or correlation with an effect or activity. TheMarker can be any characteristic or identifier, including for example, achemical, a fluid, a protein, gene, promoter, enzyme, protein, labeledmolecule, tagged molecule, antibody, and the like.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

The compounds (e.g., PDE inhibitors, additional therapeutic agents)described herein can also be any of salts, prodrugs and prodrug salts ofsaid compound, or any solvates, hydrates and polymorphs of any of theforegoing. The compounds of this invention may contain one or moreasymmetric centers and thus occur as racemates and racemic mixtures,single enantiomers, individual diastereomers and diastereomericmixtures. All such isomeric forms of these compounds are expresslyincluded in the present invention. The compounds of this invention mayalso be represented in multiple tautomeric forms, in such instances, theinvention expressly includes all tautomeric forms of the compoundsdescribed herein. All crystal forms of the compounds described hereinare expressly included in the present invention.

As used herein, the term “salt” or “pharmaceutically acceptable salt,”is a salt formed from, for example, an acid and a basic group of acompound of any one of the formulae disclosed herein. Illustrative saltsinclude, but are not limited, to sulfate, citrate, acetate, chloride,bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, besylate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, andp-toluenesulfonate salts. The term “pharmaceutically acceptable salt”also refers to a salt prepared from a compound of any one of theformulae disclosed herein having an acidic functional group, such as acarboxylic acid functional group, and a pharmaceutically acceptableinorganic or organic base. Suitable bases include, but are not limitedto, hydroxides of alkali metals such as sodium, potassium, and lithium;hydroxides of alkaline earth metal such as calcium and magnesium;hydroxides of other metals, such as aluminum and zinc; ammonia, andorganic amines, such as unsubstituted or hydroxy-substituted mono-, di-,or trialkylamines; dicyclohexylamine; tributyl amine; pyridine;N-methyl,N-ethylamine; diethylamine; triethylamine; mono-, bis-, ortris-(2-hydroxy-lower alkyl amines), such as mono-, bis-, ortris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine, ortris-(hydroxymethyl)methylamine, N, N,-di-lower alkyl-N-(hydroxy loweralkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)amine, ortri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such asarginine, lysine, and the like. The term “pharmaceutically acceptablesalt” also refers to a salt prepared from a compound of any one of theformulae disclosed herein having a basic functional group, such as anamino functional group, and a pharmaceutically acceptable inorganic ororganic acid. Suitable acids include hydrogen sulfate, citric acid,acetic acid, hydrochloric acid (HCl), hydrogen bromide (HBr), hydrogeniodide (HI), nitric acid, phosphoric acid, lactic acid, salicylic acid,tartaric acid, ascorbic acid, succinic acid, maleic acid, besylic acid,fumaric acid, gluconic acid, glucaronic acid, formic acid, benzoic acid,glutamic acid, methanesulfonic acid, ethanesulfonic acid,benzenesulfonic acid, and p-toluenesulfonic acid.

Methods of the Invention

The invention generally provides compositions comprising PDE5 inhibitorsthat are useful for the prevention or treatment of disease or diseasesymptoms (e.g., any disease delineated herein). Compositions and methodsof the invention are particularly useful for the treatment or preventionof proliferative diseases, cancer, or tumors. This invention is based,in part, on the discoveries that PDE5 is useful for treating conditionsdelineated herein.

The methods herein include administering to the subject (including asubject identified as in need of such treatment) an effective amount ofa compound described herein, or a composition described herein toproduce a beneficial effect to the subject. Identifying a subject inneed of such treatment can be in the judgment of a subject or a healthcare professional and can be subjective (e.g. opinion) or objective(e.g. measurable by a test or diagnostic method).

As used herein, the terms “treat,” treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or symptoms associatedtherewith. It will be appreciated that, although not precluded, treatinga disorder or condition does not require that the disorder, condition orsymptoms associated therewith be completely eliminated.

As used herein, the terms “prevent,” “preventing,” “prevention,”“prophylactic treatment” and the like refer to reducing the probabilityof developing a disorder or condition in a subject, who does not have,but is at risk of or susceptible to developing a disorder or condition.

The therapeutic methods of the invention (which include prophylactictreatment) in general comprise administration of a therapeuticallyeffective amount of a compound described herein, such as a PDE5inhibitor (e.g., vardenafil, tadalafil, or sildenafil) to a subject(e.g., animal, human) in need thereof, including a mammal, particularlya human. Such treatment will be suitably administered to subjects,particularly humans, suffering from, having, susceptible to, or at riskfor a disease, disorder, or symptom thereof. Determination of thosesubjects “at risk” can be made by any objective or subjectivedetermination by a diagnostic test or opinion of a subject or healthcare provider (e.g., genetic test, enzyme or protein marker, Marker (asdefined herein), family history, and the like). The compounds herein maybe also used in the treatment of any other disorders in which aremediated by an immune response (e.g., anti-tumor immune response).

In aspects of the methods herein, assays are used to monitor thecondition of a subject prior to, during, or following treatment with aPDE5A inhibitor. Treatments can be used in conjunction with one or morerelevant diagnostic test(s) for determining the efficacy, theprogression, or the appropriate dosage in the methods of the invention.

Any number of standard methods are available for assaying certainmarkers. Methods for assaying include any one or more of the following:tumor size, measurement, x-ray, CAT scan, magnetic resonance imaging,protein expression, nucleic acid expression, isotopologue, radiolabel,fluorescent probe, and the like.

Prophylactic and Therapeutic Applications

One aspect is a method of modulating myeloid suppressor cells (MSCs)immune suppression in a subject comprising administration to the subjectof a PDE-5 inhibitor compound. Another aspect is a method of modulatingarginase-1 (Arg-1) activity in a subject identified as in need of suchtreatment comprising administration to the subject of a PDE-5 inhibitorcompound. Another aspect is a method of modulating nitric oxide synthase2 (NOS2) activity in a subject identified as in need of such treatmentcomprising administration to the subject of a PDE-5 inhibitor compound.Another aspect is a method of modulating (e.g., down-regulating)interleukin-4Rα (IL-4Rα) activity in a subject identified as in need ofsuch treatment comprising administration to the subject of a PDE-5inhibitor compound. The methods can be wherein the modulating isdown-regulation.

Another aspect is a method of modulating (e.g., activating) CD8+ T cellsin a subject identified as in need of such treatment comprisingadministration to the subject of a PDE-5 inhibitor compound. Anotheraspect is a method of improving the efficacy of immune-based treatmentprotocols of malignancies in a subject comprising the step of furtheradministration to the subject of a PDE-5 inhibitor compound in additionto the immune-based treatment steps.

Another aspect is a method of reducing tumor size in a subjectcomprising administration to the subject an effective amount of a PDE-5inhibitor, wherein the subject is identified as in need of anti-tumortreatment with a PDE-5 inhibitor compound.

Another aspect is a method of modulating the suppressive function ofArg-1 or NOS2 in a subject comprising administration to the subject aneffective amount of a PDE-5 inhibitor, wherein the subject is identifiedas in need of such treatment with a PDE-5 inhibitor compound.

Another aspect is a method of reducing NO production in a subjectcomprising administration to the subject an effective amount of a PDE-5inhibitor, wherein the subject is identified as in need of suchtreatment with a PDE-5 inhibitor compound.

Another aspect is a method of modulating T-cell proliferation in asubject comprising administration to the subject an effective amount ofa PDE-5 inhibitor, wherein the subject is identified as in need of suchtreatment with a PDE-5 inhibitor compound.

Another aspect is a method of modulating the efficacy of adoptive T-cellimmunotherapy in a subject comprising administration to the T-cells aneffective amount of a PDE-5 inhibitor, wherein the administrationresults in expansion of T cells in vitro in the presence of PDE-5inhibitors. The PDE-5 inhibitor is thus useful as an adjuvant inadoptive cell transfer (ACT) protocols. Such pre-treatment of T-cellswith a PDE-5 inhibitor can enhance the effectiveness of a therapeutic(e.g., vaccine) by modulating immunosuppression when administered to asubject, thus allowing for greater efficacy of the vaccine itself.

Another aspect is a method of modulating peripheral blood lymphocyte(PBL) proliferation in a subject comprising administration to thesubject an effective amount of a PDE-5 inhibitor, wherein the subject isidentified as in need of such treatment with a PDE-5 inhibitor compound.

Another aspect is a method of modulating CD4⁺ or CD8⁺ proliferation in asubject comprising administration to the subject an effective amount ofa PDE-5 inhibitor, wherein the subject is identified as in need of suchtreatment with a PDE-5 inhibitor compound.

In other aspects, the compositions and methods for treating, preventingor modulating disease herein are those wherein the disease is a diseasemediated by myeloid suppressor cells (MSCs). The compositions andmethods for treating, preventing or modulating disease include diseasessuch as cancer (e.g., multiple myeloma, lymphomas, melanoma, breast,stomach, head and neck, ovarian, colon, prostate, lung, high gradegliomas, or cervical cancer), chronic infection, chronic inflammation,and hematopoietic reconstitution following chemotherapy. The methods canfurther comprise the step of assessing MSC levels in the subject; or canfurther comprise the steps of assessing MSC levels in the subject beforeand after administration. The assessment can be made by surface markerexpression, by MSC number, or by measure of immunosuppression function.

In each of the embodiments herein, an additional therapeutic agent maybe administered together with a PDE inhibitor compound of this inventionas part of a single dosage form or as separate dosage forms.Alternatively, the additional agent may be administered prior to,consecutively with, or following the administration of a compound ofthis invention. In such combination therapy treatment, both thecompounds of this invention and the second therapeutic agent(s) areadministered by conventional methods. The administering of the secondtherapeutic agent may occur before, concurrently with, and/or after theadministering of the compound of this invention. When administration ofthe second therapeutic agent occurs concurrently with a compound of thisinvention, the two (or more) agents may be administered in a singledosage form (such as a composition of this invention comprising acompound of the invention and an second therapeutic agent as describedabove), or in separate dosage forms. The administration of a compositionof this invention comprising both a compound of the invention and asecond therapeutic agent to a subject does not preclude the separateadministration of said second therapeutic agent, any other therapeuticagent or any compound of this invention to said subject at another timeduring a course of treatment.

Examples of additional anticancer therapeutic agents include, forexample, an antiangiogenesis agent, selective estrogen-receptormodulator (SERM), breast cancer therapeutic agent, aromatase inhibitor,biologic response modifiers, hormonal therapies agent, anthracycline,taxane, alkylating agent, taxol, cis-platin, arabinofuranosyl cytosine(ara-C), 5-fluorouracil (5-FU), altretamine, busulfan, chlorambucil,cyclophosphamide, ifosfamide, mechlorethamine, melphalan, thiotepa,cladribine, fluorouracil, floxuridine, gemcitabine, thioguanine,pentostatin, methotrexate, 6-mercaptopurine, cytarabine, carmustine,lomustine, streptozotocin, carboplatin, oxaliplatin, iproplatin,tetraplatin, lobaplatin, JM216, JM335, fludarabine, aminoglutethimide,flutamide, goserelin, leuprolide, megestrol acetate, cyproteroneacetate, tamoxifen, anastrozole, bicalutamide, dexamethasone,diethylstilbestrol, prednisone, bleomycin, dactinomycin, daunorubicin,doxirubicin, idarubicin, mitoxantrone, losoxantrone, mitomycin-c,plicamycin, paclitaxel, docetaxel, CPI-11, epothilones, topotecan,irinotecan, 9-amino camptothecan, 9-nitro camptothecan, GS-211,etoposide, teniposide, vinblastine, vincristine, vinorelbine,procarbazine, asparaginase, pegaspargase, methoxtrexate, octreotide,estramustine, hydroxyurea, tamoxifen, raloxifene, toremifene,exemestane, letrozole, anastrozole, megestrol, trastuzumab, goserelinacetate, fulvestrant, doxorubicin, epirubicin, or cyclophosphonamide andthe like Immunotherapeutic agents are also useful in the embodimentsdelineated herein. Examples of immune-based strategies includecancer-specific vaccines such as DNA-based, protein-based, whole celltumor vaccines, dendritic cell based vaccines; adoptive T cell therapy;strategies aimed at augmenting T cell function through blockade orelimination of inhibitory or suppressor mechanisms such as CTLA-4blockade, elimination of regulatory T cells (Tregs) or abrogation ofmyeloid suppressor mechanisms such as in this patent or throughactivation of T cells such as anti-CD3/CD28 stimulation, growth in IL-2.

PDE5 Inhibitors

PDE5 inhibitors are known in the art, and include, but are not limitedto, sildenafil (Compound 1), vardenafil (Compound 2), tadalafil(Compound 3), EMD 360527, DA 8159, or analogs thereof, or any othercompound that inhibits cGMP hydrolysis by phosphodiesterase-5 (PDE5).

Certain compounds useful in the present invention can be represented bythe structure (Formula I):

in which R¹ is H, C₁-C₃ alkyl, C₃-C₅ cycloalkyl or C₁-C₃ perfluoroalkyl;R² is H, C₁-C₆ alkyl optionally substituted by OH, C₁-C₃ alkoxy or C₃-C₆cycloalkyl, or C₁-C₃ perfluoroalkyl; R³ is C₁-C₆ alkyl, C₃-C₆ alkenyl,C₃-C₆ alkynyl, C₃-C₆₇ cycloalkyl, C₁-C₆ perfluoroalkyl or (C₃-C₆cycloalkyl) C₁-C₆ alkyl; R⁴ taken together with the nitrogen atom towhich it is attached completes a 4-N—(R⁶)-piperazinyl group; R⁵ is H,C₁-C₄ alkyl, C₁-C₃ alkoxy, NR⁷R⁸, or CON R⁷R⁸; R⁶ is H, C₁-C₆ alkyl,(C₁-C₃ alkoxy) C₂-C₆ alkyl hydroxy C₂-C₆ alkyl, (R⁷R⁸N) C₂-C₆ alkyl,(R⁷R⁸NCO) C₁-C₆ alkyl, CON R⁷R⁸, CSN R⁷R⁸ or C(NH)N R⁷R⁸; R⁷ and R⁸ areeach independently H, C₁-C₄ alkyl, (C₁-C₃ alkoxy) C₂-C₄ alkyl or hydroxyC₂-C₄ alkyl; and pharmaceutically acceptable salts thereof.

Other preferred compounds for use in the present invention are disclosedin U.S. Pat. No. 6,362,178 and can be represented by the structure(Formula II):

in which

R¹ represents hydrogen or straight-chain or branched alkyl having up to4 carbon atoms,

R² represents straight-chain alkyl having up to 4 carbon atoms,

R³ and R⁴ are identical or different and each represents hydrogen orrepresents straight-chain or branched alkenyl or alkoxy having in eachcase up to 8 carbon atoms, or represents a straight-chain or branchedalkyl chain having up to 10 carbon atoms which is optionally interruptedby an oxygen atom and which is optionally mono- or polysubstituted byidentical or different substituents selected from the group consistingof trifluoromethyl, trifluoromethoxy, hydroxyl, halogen, carboxyl,benzyloxycarbonyl, straight-chain or branched alkoxycarbonyl having upto 6 carbon atoms and/or by radicals of the formulae —SO₃H,-(A)_(a)-NR⁷R⁸, —O—CO—NR^(7′)R^(8′), —S(O)_(b)—R⁹, —P(O)(OR¹⁰)(OR¹¹),

in which

a and b are identical or different and each represents a number 0 or 1,

A represents a radical CO or SO₂,

R⁷, R^(7′), R⁸ and R^(8′) are identical or different and each representshydrogen, or represents cycloalkyl having 3 to 8 carbon atoms, arylhaving 6 to 10 carbon atoms, a 5- to 6-membered unsaturated, partiallyunsaturated or saturated, optionally benzo-fused heterocycle having upto 3 heteroatoms from the group consisting of S, N and O, where theabovementioned ring systems are optionally mono- or polysubstituted byidentical or different substituents selected from the group consistingof hydroxyl, nitro, trifluoromethyl, trifluoromethoxy, carboxyl,halogen, straight-chain or branched alkoxy or alkoxycarbonyl having ineach case up to 6 carbon atoms or by a group of the formula—(SO₂)_(c)—NR¹²R¹³, in which

c represents a number 0 or 1,

R¹² and R¹³ are identical or different and each represents hydrogen orstraight-chain or branched alkyl having up to 5 carbon atoms, or

R⁷, R^(7′), R⁸ and R^(8′) each represent straight-chain or branchedalkoxy having up to 6 carbon atoms, or represents straight-chain orbranched alkyl having up to 8 carbon atoms which is optionally mono- orpolysubstituted by identical or different substituents selected from thegroup consisting of hydroxyl, halogen, aryl having 6 to 10 carbon atoms,straight-chain or branched alkoxy or alkoxycarbonyl having in each caseup to 6 carbon atoms or by a group of the formula —(CO)_(d)—NR¹⁴R¹⁵, inwhich

R¹⁴ and R¹⁵ are identical or different and each represents hydrogen orstraight-chain or branched alkyl having up to 4 carbon atoms, and

d represents a number 0 or 1, or

R⁷ and R⁸ and/or R^(7′) and R^(8′) together with the nitrogen atom forma 5- to 7-membered saturated heterocycle which may optionally contain afurther heteroatom from the group consisting of S and O or a radical ofthe formula —NR¹⁶, in which

R¹⁶ represents hydrogen, aryl having 6 to 10 carbon atoms, benzyl, a 5-to 7-membered aromatic or saturated heterocycle having up to 3heteroatoms from the group consisting of S, N and O which is optionallysubstituted by methyl, or represents straight-chain or branched alkylhaving up to 6 carbon atoms which is optionally substituted by hydroxyl,

R⁹ represents aryl having 6 to 10 carbon atoms, or representsstraight-chain or branched alkyl having up to 4 carbon atoms,

R¹⁰ and R¹¹ are identical or different and each represents hydrogen orstraight-chain or branched alkyl having up to 4 carbon atoms, and/or thealkyl chain listed above under R³/R⁴ is optionally substituted bycycloalkyl having 3 to 8 carbon atoms, aryl having 6 to 10 carbon atomsor by a 5- to 7-membered partially unsaturated, saturated orunsaturated, optionally benzo-fused heterocycle which may contain up to4 heteroatoms from the group consisting of S, N and O or a radical ofthe formula —NR¹⁷, in which

R¹⁷ represents hydrogen, hydroxyl, formyl, trifluoromethyl,straight-chain or branched acyl or alkoxy having in each case up to 4carbon atoms, or represents straight-chain or branched alkyl having upto 6 carbon atoms which is optionally mono- or polysubstituted byidentical or different substituents selected from the group consistingof hydroxyl and straight-chain or branched alkoxy having up to 6 carbonatoms, and where aryl and the heterocycle are optionally mono- orpolysubstituted by identical or different substituents selected from thegroup consisting of nitro, halogen, —SO₃H, straight-chain or branchedalkyl or alkoxy having in each case up to 6 carbon atoms, hydroxyl,trifluoromethyl, trifluoromethoxy and/or by a radical of the formula—SO₂—NR¹⁸R¹⁹, in which

R¹⁸ and R¹⁹ are identical or different and each represents hydrogen orstraight-chain or branched alkyl having up to 6 carbon atoms, and/or

R³ or R⁴ represents a group of the formula —NR²⁰R²¹, in which R²⁰ andR²¹ have the meanings of R¹⁸ and R¹⁹ given above and are identical to ordifferent from them, and/or

R³ or R⁴ represents adamantyl, or represents radicals of the formulae

or represents cycloalkyl having 3 to 8 carbon atoms, aryl having 6 to 10carbon atoms or represents a 5- to 7-membered partially unsaturated,saturated or unsaturated, optionally benzo-fused heterocycle which maycontain up to 4 heteroatoms from the group consisting of S, N and O, ora radical of the formula —NR²², in which

R²² has the meaning of R¹⁶ given above and is identical to or differentfrom it, or represents carboxyl, formyl or straight-chain or branchedacyl having up to 5 carbon atoms, and where cycloalkyl, aryl and/or theheterocycle are optionally mono- or polysubstituted by identical ordifferent substituents selected from the group consisting of halogen,triazolyl, trifluoromethyl, trifluoromethoxy, carboxyl, straight-chainor branched acyl or alkoxycarbonyl having in each case up to 6 carbonatoms, nitro and/or by groups of the formulae —SO₃H, —OR²³,(SO₂)_(c)NR²⁴R²⁵, —P(O)(OR²⁶)(OR²⁷), in which

e represents a number 0 or 1,

R²³ represents a radical of the formula

orrepresents cycloalkyl having 3 to 7 carbon atoms, or represents hydrogenor straight-chain or branched alkyl having up to 4 carbon atoms which isoptionally substituted by cycloalkyl having 3 to 7 carbon atoms,benzyloxy, tetrahydropyranyl, tetrahydrofuranyl, straight-chain orbranched alkoxy or alkoxycarbonyl having in each case up to 6 carbonatoms, carboxyl, benzyloxycarbonyl or phenyl which for its part may bemono- or polysubstituted by identical or different substituents selectedfrom the group consisting of straight-chain or branched alkoxy having upto 4 carbon atoms, hydroxyl and halogen, and/or alkyl which isoptionally substituted by radicals of the formulae —CO—NR²⁸R²⁹ or—CO—R³⁰, in which

R²⁸ and R²⁹ are identical or different and each represents hydrogen orstraight-chain or branched alkyl having up to 8 carbon atoms, or

R²⁸ and R²⁹ together with the nitrogen atom form a 5- to 7-memberedsaturated heterocycle which may optionally contain a further heteroatomfrom the group consisting of S and O, and

R³⁰ represents phenyl or adamantyl,

R²⁴ and R²⁵ have the meanings of R¹⁸ and R¹⁹ given above and areidentical to or different from them,

R²⁶ and R²⁷ have the meanings of R¹⁰ and R¹¹ given above and areidentical to or different from them and/or cycloalkyl, aryl and/or theheterocycle are optionally substituted by straight-chain or branchedalkyl having up to 6 carbon atoms which is optionally substituted byhydroxyl, carboxyl, by a 5- to 7-membered heterocycle having up to 3heteroatoms from the group consisting of S, N and O, or by groups of theformula —SO₂—R³¹, P(O)(OR³²)(OR³³) or —NR³⁴R³⁵, in which

R³¹ represents hydrogen or has the meaning of R⁹ given above and isidentical to or different from it,

R³² and R³³ have the meanings of R¹⁰ and R¹¹ given above and areidentical to or different from them,

R³⁴ and R³⁵ are identical or different and each represents hydrogen orstraight-chain or branched alkyl having up to 6 carbon atoms which isoptionally substituted by hydroxyl or by straight-chain or branchedalkoxy having up to 4 carbon atoms, or

R³⁴ and R³⁵ together with the nitrogen atom form a 5- to 6-memberedsaturated heterocycle which may contain a further heteroatom from thegroup consisting of S and O, or a radical of the formula —NR³⁶, in which

R³⁶ represents hydrogen, hydroxyl, straight-chain or branchedalkoxycarbonyl having up to 7 carbon atoms or straight-chain or branchedalkyl having up to 5 carbon atoms which is optionally substituted byhydroxyl, or

R³ and R⁴ together with the nitrogen atom form a 5- to 7-memberedunsaturated or saturated or partially unsaturated, optionallybenzo-fused heterocycle which may optionally contain up to 3 heteroatomsfrom the group consisting of S, N and O, or a radical of the formula—NR³⁷, in which

R³⁷ represents hydrogen, hydroxyl, formyl, trifluoromethyl,straight-chain or branched acyl, alkoxy or alkoxycarbonyl having in eachcase up to 4 carbon atoms, or represents straight-chain or branchedalkyl having up to 6 carbon atoms which is optionally mono- orpolysubstituted by identical or different substituents selected from thegroup consisting of hydroxyl, trifluoromethyl, carboxyl, straight-chainor branched alkoxy or alkoxycarbonyl having in each case up to 6 carbonatoms, or by groups of the formula -(D)_(f)-NR³⁸R³⁹,—CO—(CH₂)_(g)—O—CO—R⁴⁰, —CO—(CH₂)_(h)—OR⁴¹ or —P(O)(OR⁴²)(OR⁴³), inwhich

g and h are identical or different and each represents a number 1, 2, 3or 4, and

f represents a number 0 or 1,

D represents a group of the formula —CO or —SO₂,

R³⁸ and R³⁹ are identical or different and each has the meaning of R⁷and R⁸ given above,

R⁴⁰ represents straight-chain or branched alkyl having up to 6 carbonatoms,

R⁴¹ represents straight-chain or branched alkyl having up to 6 carbonatoms,

R⁴² and R⁴³ are identical or different and each represents hydrogen orstraight-chain or branched alkyl having up to 4 carbon atoms, or

R³⁷ represents a radical of the formula —(CO)_(i)-E, in which irepresents a number 0 or 1,

E represents cycloalkyl having 3 to 7 carbon atoms or benzyl, representsaryl having 6 to 10 carbon atoms or a 5- to 6-membered aromaticheterocycle having up to 4 heteroatoms from the group consisting of S, Nand O, where the abovementioned ring systems are optionally mono- orpolysubstituted by identical or different constituents selected from thegroup consisting of nitro, halogen, —SO₃H, straight-chain or branchedalkoxy having up to 6 carbon atoms, hydroxyl, trifluoromethyl,trifluoromethoxy, or by a radical of the formula —SO₂—NR⁴⁴R⁴⁵, in which

R⁴⁴ and R⁴⁵ have the meaning of R¹⁸ and R¹⁹ given above and areidentical to or different from them, or

E represents radicals of the formulae

and the heterocycle listed under R³ and R⁴, which is formed togetherwith the nitrogen atom, is optionally mono- or polysubstituted, ifappropriate also geminally, by identical or different substituentsselected from the group consisting of hydroxyl, formyl, carboxyl,straight-chain or branched acyl or alkoxycarbonyl having in each case upto 6 carbon atoms, nitro and groups of the formulae —P(O)(OR⁴⁶)(OR⁴⁷),

═NR⁴⁸, or —C(O)_(j)NR⁴⁹R⁵⁰,in which

R⁴⁶ and R⁴⁷ have the meanings of R¹⁰ and R¹¹ given above and areidentical to or different from them,

R⁴⁸ represents hydroxyl or straight-chain or branched alkoxy having upto 4 carbon atoms, j represents a number 0 or 1, and

R⁴⁹ and R⁵⁰ are identical or different and have the meanings of R¹⁴ andR¹⁵ given above, and/or the heterocycle listed under R³ and R⁴, which isformed together with the nitrogen atom, is optionally substituted bystraight-chain or branched alkyl having up to 6 carbon atoms which isoptionally mono- or polysubstituted by identical or differentsubstituents selected from the group consisting of hydroxyl, halogen,carboxyl, cycloalkyl or cycloalkyloxy having in each case 3 to 8 carbonatoms, straight-chain or branched alkoxy or alkoxycarbonyl having ineach case up to 6 carbon atoms, or by a radical of the formula —SO₃H,—NR⁵¹R⁵² or P(O)OR⁵³OR⁵⁴, in which

R⁵¹ and R⁵² are identical or different and each represents hydrogen,phenyl, carboxyl, benzyl or straight-chain or branched alkyl or alkoxyhaving in each case up to 6 carbon atoms,

R⁵³ and R⁵⁴ are identical or different and have the meanings of R¹⁰ andR¹¹ given above, and/or the alkyl is optionally substituted by arylhaving 6 to 10 carbon atoms which for its part may be mono- orpolysubstituted by identical or different substituents selected from thegroup consisting of halogen, hydroxyl, straight-chain or branched alkoxyhaving up to 6 carbon atoms, or by a group of the formula—NR^(51′)R^(52′), in which

R^(51′) and R^(52′) have the meanings of R⁵¹ and R⁵² given above and areidentical to or different from them, and/or the heterocycle listed underR³ and R⁴, which is formed together with the nitrogen atom, isoptionally substituted by aryl having 6 to 10 carbon atoms or by a 5- to7-membered saturated, partially unsaturated or unsaturated heterocyclehaving up to 3 heteroatoms from the group consisting of S, N and O,optionally also attached via a nitrogen function, where the ring systemsfor their part may be substituted by hydroxyl or by straight-chain orbranched alkyl or alkoxy having in each case up to 6 carbon atoms, or

R³ and R⁴ together with the nitrogen atom form radicals of the formulae

R⁵ and R⁶ are identical or different and each represents hydrogen,straight-chain or branched alkyl having up to 6 carbon atoms, hydroxylor represents straight-chain or branched alkoxy having up to 6 carbonatoms,and their salts, hydrates, N-oxides and structural isomers.

Other suitable compounds include those of the following Formula III:

wherein in Formula III, R⁰ represents hydrogen, halogen, or C₁₋₆ alkyl;

R¹ represents hydrogen, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, haloC₁₋₆alkyl, C₃₋₈ cycloalkyl, C₃₋₈ cycloalkylC₁₋₃ alkyl, arylC₁₋₃ alkyl, orheteroarylC₁₋₃ alkyl;

R² represents an optionally substituted monocyclic aromatic ringselected from benzene, thiophene, furan, and pyridine, or an optionallysubstituted bicyclic ring;

attached to the rest of the molecule via one of the benzene ring carbonatoms and wherein the fused ring A is a 5- or 6-membered ring which maybe saturated or partially or fully unsaturated and comprises carbonatoms and optionally one or two heteroatoms selected from oxygen,sulphur, and nitrogen; and

R³ represents hydrogen of C₁₋₃ alkyl, or R¹ and R³ together represent a3- or 4-membered alkyl or alkenyl chain; and pharmaceutically and saltsand solvates (e.g., hydrates) thereof.

Certain preferred compounds also include those of the following FormulaIV:

wherein in Formula IV, R⁰ represents hydrogen, halogen, or C₁₋₆ alkyl;

R¹ represents hydrogen, C₁₋₆ alkyl, haloC₁₋₆ alkyl, C₃₋₈ cycloalkylC₁₋₃alkyl, arylC₁₋₃ alkyl, or heteroarylC₁₋₃ alkyl; andR² represents an optionally substituted monocyclic aromatic ringselected from benzene, thiophene, furan, and pyridine, or an optionallysubstituted bicyclic ring

attached to the rest of the molecule via one of the benzene ring carbonatoms, and wherein the fused ring A is a 5- or 6-membered ring which canbe saturated or partially or fully unsaturated and comprises carbonatoms and optionally one or two heteroatoms selected from oxygen,sulphur, and nitrogen; and pharmaceutically acceptable salts andsolvates (e.g., hydrates) thereof.

A further group of compounds preferred for use in the invention arecompounds of the following Formula V:

wherein in Formula V:R⁰ represents hydrogen, halogen, or C₁₋₆ alkyl;R¹ represents hydrogen or C₁₋₆ alkyl;R² represents the bicyclic ring

which can be optionally substituted by one or more groups selected fromhalogen and C₁₋₃ alkyl; and

R³ represents hydrogen or C₁₋₃ alkyl; and pharmaceutically acceptablesalts and solvates (e.g., hydrates) thereof.

In Formula IV above, with respect to R¹, the term “aryl” as part of anarylC₁₋₃ alkyl group means phenyl or phenyl substituted by one or more(e.g., 1, 2, or 3) substituents selected from halogen, C₁₋₆ alkyl, C₁₋₆alkoxy, and methylenedioxy. The term “heteroaryl” as part of aheteroarylC₁₋₃ alkyl group means thienyl, furyl, or pyridyl, eachoptionally substituted by one or more (e.g., 1, 2, or 3) substituentsselected from halogen, C₁₋₆ alkyl, and C₁₋₆ alkoxy. The term “C₃₋₈cycloalkyl” as a group or part of a C₃₋₈ cycloalkylC₁₋₃ alkyl groupmeans a monocyclic ring comprising three to eight carbon atoms. Examplesof suitable cycloalkyl rings include the C₃₋₆ cycloalkyl ringscyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

In formula IV above, with respect to R², optional benzene ringsubstituents are selected from one or more (e.g., 1, 2, or 3) atoms orgroups comprising halogen, hydroxy, C₁₋₆ alkyl, C₁₋₆ alkoxy, CO₂ R^(b),haloC₁₋₆ alkyl, haloC₁₋₆ alkoxy, cyano, nitro, and NR^(a)R^(b), whereR^(a) and R^(b) are each hydrogen or C₁₋₆ alkyl, or R^(a) also canrepresent C₂₋₇ alkanoyl or C₁₋₆ alkylsulphonyl. Optional substituentsfor the remaining ring systems are selected from one or more (e.g., 1,2, or 3 atoms or groups comprising halogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, andarylC₁₋₃ alkyl as defined above. The bicyclic ring

can, for example, represent naphthalene, a heterocycle such asbenzoxazole, benzothiazole, benzisoxazole, benzimidazole, quinoline,indole, benzothiophene, benzofuran, or

wherein n is an integer 1 or 2 and X and Y each can represent CH₂, O, S,or NH.

See also U.S. Pat. Nos. 6,916,927, 6,911,542, 6,903,099, 6,878,711,6,872,721, 6,858,620, 6,825,197, 6,774,128, 6,723,719, 6,699,870,6,670,366, 5,859,006 and 5,250,534. Other PDE5 inhibitors useful in themethods of the invention are described in WO 03/063875; WO 03/1012761 WO2004/037183, and WO 98/38168. All of these patents and patentapplications are incorporated herein by reference in their entirety.

Sildenafil is commercially available in three dosages of 25, 50, or 100mg and has an IC₅₀ of approximately 10 nM. Effective plasmaconcentrations are between 1 nM and 250 nM, where the bottom of therange is any integer between 1 and 249; and the top of the range is anyinteger between 2 nM and 250 nM. Preferably, an effective plasmaconcentration is between 5 nM and 100 nM, more preferably it is between10 nM and 50 nM (e.g., 15 nM, 20 nM, 25 nM, 30 nM, 40 nM, or 45 nM).

Tadalafil is commercially available in three dosages of 5, 10, or 20 mgand has an IC₅₀ of approximately 1 nM. Following oral administration ofa 20 mg dose of tadalafil to healthy subjects, tadalafil is rapidlyabsorbed with the peak plasma concentration of 378 ng/ml occurring twohours post-dose. Preferably an effective plasma concentration is between5 nM and 100 nM, more preferably it is between 10 nM and 50 nM (e.g., 15nM, 20 nM, 25 nM, 30 nM, 40 nM, or 45 nM). Tadalafil has a relativelarge apparent volume of distribution (Vd/F) of 62.6 L, and a lowapparent oral clearance (CL/F) of 2.48 L/h. As a result, the meanelimination half-life of tadalafil is about 17.5 h, which issubstantially longer than that of sildenafil or vardenafil.

Vardenafil is commercially available in three dosages of 5 mg, 10 mg,and 20 mg and has an IC₅₀ of 0.7 nM. Effective plasma concentrations ofvardenafil are between 0.1 and 5.0 nM.

The skilled artisan appreciates that any compound that reduces theactivity of PDE5 is useful in the methods of the invention. Otherexemplary compounds useful in the methods of the invention includeUK-343,664 (Walker et al., Xenobiotica, 31: 651-664), UK-427,387,UK-357903[1-ethyl-4-{3-[3-ethyl-6,7-dihydro-7-oxo-2-(2-pyridylmethyl)-2H-pyrazolo[4,3-d]pyrimidin-5-yl]-2-(2-methoxyethoxy)-5-pyridylsulphonyl}piperazine](Gardiner et al. J Pharmacol Exp Ther. 2005; 312: 265-271), UK-371800(Pfizer), UK-313794 (Pfizer) and UK-343664 (Abel et al., Xenobiotica.2001 31:665-76); TA-1790 from Tanabe Seiyaku; CP-248, CP-461 andexisulind (Deguchi et al., Molecular Cancer Therapeutics 803-809, 2002),which are available from Osi Pharmaceuticals; pyrazolinone; EMD82639(4-(4-[2-ethyl-phenylamino)-methylene]-3-methyl-5-oxo-4,5-di-hydro-pyrazol-1-yl)-benzoicacid (Senzaki et al., FASEB Journal. 2001; 15:1718-1726);[7-(3-Chloro-4-methoxy-benzylamino)-1-methyl-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-5-ylmethoxyl]-aceticacid (EMD360527),4-[4-(3-Chloro-4-methoxy-benzylamino)-benzo[4,5]thieno[2,3-d]-pyrimidin-2-yl]-cyclohexanecarboxylicacid, ethanolamin salt (EMD221829) and5-[4-(3-Chloro-4-methoxy-benzylamino)-5,6,7,8-tetrahydro-benzo[4,5]thieno[2,3-d]pyrimidin-2-yl]-pentanoicacid (EMD171827), which are commercially available from Merck KgaA(Damistadt, DE) and are described, for example, in Scutt et al. (BMCPharmacol. 2004; 4: 10);3-(1-Methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo-[4,3-d]pyrimidin-5-yl)-N-[2-(1-methylpyrrolidin-2-yl)ethyl]-4-propoxybenzenesulfonamide(DA-8259); E-4021 (Dukarm et al., Am. J. Respir. Crit. Care Med., 1999,160:858-865); pentoxifylline and FR22934 (Fujisawa).

The synthesized compounds can be separated from a reaction mixture andfurther purified by a method such as column chromatography, highpressure liquid chromatography, or recrystallization. As can beappreciated by the skilled artisan, methods of synthesizing thecompounds of the formulae herein will be evident to those of ordinaryskill in the art. Additionally, the various synthetic steps may beperformed in an alternate sequence or order to give the desiredcompounds. Synthetic chemistry transformations and protecting groupmethodologies (protection and deprotection) useful in synthesizing thecompounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, 2nd. Ed., Wiley-VCH Publishers (1999); T. W. Greene andP. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd. Ed., JohnWiley and Sons (1999); L. Fieser and M. Fieser, Fieser and Fieser'sReagents for Organic Synthesis, John Wiley and Sons (1999); and L.Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, JohnWiley and Sons (1995), and subsequent editions thereof.

Pharmaceutical Compositions

The present invention features pharmaceutical preparations comprising aPDE5 inhibitor (e.g., sildenafil, vardenafil, tadalafil, or analogsthereof) together with pharmaceutically acceptable carriers, where thecompounds provide for the treatment of disease or disease symptoms(e.g., cancer, any disease delineated herein). Pharmaceuticalpreparations of the invention have both therapeutic and prophylacticapplications. In one embodiment, a pharmaceutical composition includesan effective amount of a PDE5 inhibitor. The compositions should besterile and contain a therapeutically effective amount of a PDE5inhibitor in a unit of weight or volume suitable for administration to asubject (e.g., a human patient). The compositions and combinations ofthe invention can be part of a pharmaceutical pack, where the PDE5inhibitor is present in individual dosage amounts.

Pharmaceutical compositions of the invention to be used for prophylacticor therapeutic administration should be sterile. Sterility is readilyaccomplished by filtration through sterile filtration membranes (e.g.,0.2 μm membranes), by gamma irradiation, or any other suitable meansknown to those skilled in the art. Therapeutic compositions generallyare placed into a container having a sterile access port, for example,an intravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle. These compositions ordinarily will bestored in unit or multi-dose containers, for example, sealed ampoules orvials, as an aqueous solution or as a lyophilized formulation forreconstitution.

A PDE5 inhibitor may be combined, optionally, with a pharmaceuticallyacceptable excipient. The term “pharmaceutically-acceptable excipient”as used herein means one or more compatible solid or liquid filler,diluents or encapsulating substances that are suitable foradministration into a human. The term “carrier” denotes an organic orinorganic ingredient, natural or synthetic, with which the activeingredient is combined to facilitate administration. The components ofthe pharmaceutical compositions also are capable of being co-mingledwith a PDE5 inhibitor of the present invention, and with each other, ina manner such that there is no interaction that would substantiallyimpair the desired pharmaceutical efficacy.

Compounds of the present invention can be contained in apharmaceutically acceptable excipient. The excipient preferably containsminor amounts of additives such as substances that enhance isotonicityand chemical stability. Such materials are non-toxic to recipients atthe dosages and concentrations employed, and include buffers such asphosphate, citrate, succinate, acetate, lactate, tartrate, and otherorganic acids or their salts; tris-hydroxymethylaminomethane (TRIS),bicarbonate, carbonate, and other organic bases and their salts;antioxidants, such as ascorbic acid; low molecular weight (for example,less than about ten residues) polypeptides, e.g., polyarginine,polylysine, polyglutamate and polyaspartate; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers, such aspolyvinylpyrrolidone (PVP), polypropylene glycols (PPGs), andpolyethylene glycols (PEGs); amino acids, such as glycine, glutamicacid, aspartic acid, histidine, lysine, or arginine; monosaccharides,disaccharides, and other carbohydrates including cellulose or itsderivatives, glucose, mannose, sucrose, dextrins or sulfatedcarbohydrate derivatives, such as heparin, chondroitin sulfate ordextran sulfate; polyvalent metal ions, such as divalent metal ionsincluding calcium ions, magnesium ions and manganese ions; chelatingagents, such as ethylenediamine tetraacetic acid (EDTA); sugar alcohols,such as mannitol or sorbitol; counterions, such as sodium or ammonium;and/or nonionic surfactants, such as polysorbates or poloxamers. Otheradditives may be included, such as stabilizers, anti-microbials, inertgases, fluid and nutrient replenishers (i.e., Ringer's dextrose),electrolyte replenishers, and the like, which can be present inconventional amounts.

The compositions, as described above, can be administered in effectiveamounts. The effective amount will depend upon the mode ofadministration, the particular condition being treated and the desiredoutcome. It may also depend upon the stage of the condition, the age andphysical condition of the subject, the nature of concurrent therapy, ifany, and like factors well known to the medical practitioner. Fortherapeutic applications, it is that amount sufficient to achieve amedically desirable result.

With respect to a subject having a disease or disorder delineatedherein, an effective amount is an amount sufficient to stabilize, slow,or reduce a symptom associated with the condition. Generally, doses ofthe compounds of the present invention would be from about 0.01 mg/kgper day to about 1000 mg/kg per day. In one embodiment, 25, 50, 75, 100,125, 150 or 200 mg of a PDE5 inhibitor, such as sildenafil, isadministered to a subject. Preferably, 100 mg of a PDE5 inhibitor isadministered. Effective doses range from 0.1 nM to 200 nM, where thebottom of the range is any integer between 1 and 199, and the top of therange is any integer between 2 and 200. It is expected that dosesranging from about 5 to about 2000 mg/kg will be suitable—depending onthe specific PDE5 inhibitor used. Lower doses will result from certainforms of administration, such as intravenous administration andpharmaceutical. In the event that a response in a subject isinsufficient at the initial doses applied, higher doses (or effectivelyhigher doses by a different, more localized delivery route) may beemployed to the extent that patient tolerance permits. Multiple dosesper day are contemplated to achieve appropriate systemic levels of acomposition of the present invention.

A variety of administration routes are available. The methods of theinvention, generally speaking, may be practiced using any mode ofadministration that is medically acceptable, meaning any mode thatproduces effective levels of the active compounds without causingclinically unacceptable adverse effects. In one preferred embodiment, acomposition of the invention is administered orally. Other modes ofadministration include rectal, topical, intraocular, buccal,intravaginal, intracisternal, intracerebroventricular, intratracheal,nasal, transdermal, within/on implants, or parenteral routes or possiblyintratumorally. The term “parenteral” includes subcutaneous,intrathecal, intravenous, intramuscular, intraperitoneal, or infusion.Intravenous or intramuscular routes are not particularly suitable forlong-term therapy and prophylaxis. They could, however, be preferred inemergency situations. Compositions comprising a composition of theinvention can be added to a physiological fluid, such as blood. Oraladministration can be preferred for prophylactic treatment because ofthe convenience to the patient as well as the dosing schedule.

Pharmaceutical compositions of the invention can comprise one or more pHbuffering compounds to maintain the pH of the formulation at apredetermined level that reflects physiological pH, such as in the rangeof about 5.0 to about 8.0. The pH buffering compound used in the aqueousliquid formulation can be an amino acid or mixture of amino acids, suchas histidine or a mixture of amino acids such as histidine and glycine.Alternatively, the pH buffering compound is preferably an agent whichmaintains the pH of the formulation at a predetermined level, such as inthe range of about 5.0 to about 8.0, and which does not chelate calciumions. Illustrative examples of such pH buffering compounds include, butare not limited to, imidazole and acetate ions. The pH bufferingcompound may be present in any amount suitable to maintain the pH of theformulation at a predetermined level.

Pharmaceutical compositions of the invention can also contain one ormore osmotic modulating agents, i.e., a compound that modulates theosmotic properties (e.g, tonicity, osmolality and/or osmotic pressure)of the formulation to a level that is acceptable to the blood stream andblood cells of recipient individuals. The osmotic modulating agent canbe an agent that does not chelate calcium ions. The osmotic modulatingagent can be any compound known or available to those skilled in the artthat modulates the osmotic properties of the formulation. One skilled inthe art may empirically determine the suitability of a given osmoticmodulating agent for use in the inventive formulation. Illustrativeexamples of suitable types of osmotic modulating agents include, but arenot limited to: salts, such as sodium chloride and sodium acetate;sugars, such as sucrose, dextrose, and mannitol; amino acids, such asglycine; and mixtures of one or more of these agents and/or types ofagents. The osmotic modulating agent(s) may be present in anyconcentration sufficient to modulate the osmotic properties of theformulation.

Pharmaceutical compositions of the invention can also be a non-aqueousliquid formulation. Any suitable non-aqueous liquid may be employed,provided that it provides stability to the active agents (s) containedtherein. Preferably, the non-aqueous liquid is a hydrophilic liquid.Illustrative examples of suitable non-aqueous liquids include: glycerol;dimethyl sulfoxide (DMSO); polydimethylsiloxane (PMS); ethylene glycols,such as ethylene glycol, diethylene glycol, triethylene glycol,polyethylene glycol (“PEG”) 200, PEG 300, and PEG 400; and propyleneglycols, such as dipropylene glycol, tripropylene glycol, polypropyleneglycol (“PPG”) 425, PPG 725, PPG 1000, PPG 2000, PPG 3000 and PPG 4000.

Pharmaceutical compositions of the invention can also be a mixedaqueous/non-aqueous liquid formulation. Any suitable non-aqueous liquidformulation, such as those described above, can be employed along withany aqueous liquid formulation, such as those described above, providedthat the mixed aqueous/non-aqueous liquid formulation provides stabilityto the compound contained therein. Preferably, the non-aqueous liquid insuch a formulation is a hydrophilic liquid. Illustrative examples ofsuitable non-aqueous liquids include: glycerol; DMSO; PMS; ethyleneglycols, such as PEG 200, PEG 300, and PEG 400; and propylene glycols,such as PPG 425, PPG 725, PPG 1000, PPG 2000, PPG 3000 and PPG 4000.

Suitable stable formulations can permit storage of the active agents ina frozen or an unfrozen liquid state. Stable liquid formulations can bestored at a temperature of at least −70° C., but can also be stored athigher temperatures of at least 0° C., or between about 0.1° C. andabout 42° C., depending on the properties of the composition.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of compositions of the invention, increasing convenienceto the subject and the physician. Many types of release delivery systemsare available and known to those of ordinary skill in the art. Theyinclude polymer base systems such as polylactides (U.S. Pat. No.3,773,919; European Patent No. 58,481), poly(lactide-glycolide),copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters,polyhydroxybutyric acids, such as poly-D-(−)-3-hydroxybutyric acid(European Patent No. 133, 988), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman, K. R. et al., Biopolymers 22: 547-556),poly (2-hydroxyethyl methacrylate) or ethylene vinyl acetate (Langer, R.et al., J. Biomed. Mater. Res. 15:267-277; Langer, R. Chem. Tech.12:98-105), and polyanhydrides.

Other examples of sustained-release compositions include semi-permeablepolymer matrices in the form of shaped articles, e.g., films, ormicrocapsules. Delivery systems also include non-polymer systems thatare: lipids including sterols such as cholesterol, cholesterol estersand fatty acids or neutral fats such as mono- di- and tri-glycerides;hydrogel release systems such as biologically-derived bioresorbablehydrogel (i.e., chitin hydrogels or chitosan hydrogels); sylasticsystems; peptide based systems; wax coatings; compressed tablets usingconventional binders and excipients; partially fused implants; and thelike. Specific examples include, but are not limited to: (a) erosionalsystems in which the agent is contained in a form within a matrix suchas those described in U.S. Pat. Nos. 4,452,775, 4,667,014, 4,748,034 and5,239,660 and (b) diffusional systems in which an active componentpermeates at a controlled rate from a polymer such as described in U.S.Pat. Nos. 3,832,253, and 3,854,480.

Another type of delivery system that can be used with the methods andcompositions of the invention is a colloidal dispersion system.Colloidal dispersion systems include lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes.Liposomes are artificial membrane vessels, which are useful as adelivery vector in vivo or in vitro.

Liposomes can be targeted to a particular tissue by coupling theliposome to a specific ligand such as a monoclonal antibody, sugar,glycolipid, or protein. Liposomes are commercially available from GibcoBRL, for example, as LIPOFECTIN™ and LIPOFECTACE™, which are formed ofcationic lipids such as N-[1-(2, 3 dioleyloxy)-propyl]-N, N,N-trimethylammonium chloride (DOTMA) and dimethyl dioctadecylammoniumbromide (DDAB). Methods for making liposomes are well known in the artand have been described in many publications, for example, in DE3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692(1985); Hwang et al., Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980);EP 52,322; EP 36,676; EP 88, 046; EP 143,949; EP 142,641; Japanese Pat.Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.Liposomes also have been reviewed by Gregoriadis, G., TrendsBiotechnol., 3: 235-241).

Another type of vehicle is a biocompatible microparticle or implant thatis suitable for implantation into a mammalian recipient. Exemplarybioerodible implants that are useful in accordance with this method aredescribed in PCT International application no. PCT/US/03307 (PublicationNo. WO 95/24929, entitled “Polymeric Gene Delivery System”). PCT/US/0307describes biocompatible, preferably biodegradable polymeric matrices forcontaining an exogenous gene under the control of an appropriatepromoter. The polymeric matrices can be used to achieve sustainedrelease of the exogenous gene or gene product in the subject.

The polymeric matrix preferably is in the form of a microparticle suchas a microsphere (wherein an agent is dispersed throughout a solidpolymeric matrix) or a microcapsule (wherein an agent is stored in thecore of a polymeric shell). Microcapsules of the foregoing polymerscontaining drugs are described in, for example, U.S. Pat. No. 5,075,109.Other forms of the polymeric matrix for containing an agent includefilms, coatings, gels, implants, and stents. The size and composition ofthe polymeric matrix device is selected to result in favorable releasekinetics in the tissue into which the matrix is introduced. The size ofthe polymeric matrix further is selected according to the method ofdelivery that is to be used. Preferably, when an aerosol route is usedthe polymeric matrix and composition are encompassed in a surfactantvehicle. The polymeric matrix composition can be selected to have bothfavorable degradation rates and also to be formed of a material, whichis a bioadhesive, to further increase the effectiveness of transfer. Thematrix composition also can be selected not to degrade, but rather torelease by diffusion over an extended period of time. The deliverysystem can also be a biocompatible microsphere that is suitable forlocal, site-specific delivery. Such microspheres are disclosed inChickering, D. E., et al., Biotechnol. Bioeng., 52: 96-101; Mathiowitz,E., et al., Nature 386: 410-414.

Both non-biodegradable and biodegradable polymeric matrices can be usedto deliver the compositions of the invention to the subject. Suchpolymers may be natural or synthetic polymers. The polymer is selectedbased on the period of time over which release is desired, generally inthe order of a few hours to a year or longer. Typically, release over aperiod ranging from between a few hours and three to twelve months ismost desirable. The polymer optionally is in the form of a hydrogel thatcan absorb up to about 90% of its weight in water and further,optionally is cross-linked with multivalent ions or other polymers.

Exemplary synthetic polymers which can be used to form the biodegradabledelivery system include: polyamides, polycarbonates, polyalkylenes,polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates,polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly-vinylhalides, polyvinylpyrrolidone, polyglycolides, polysiloxanes,polyurethanes and co-polymers thereof, alkyl cellulose, hydroxyalkylcelluloses, cellulose ethers, cellulose esters, nitro celluloses,polymers of acrylic and methacrylic esters, methyl cellulose, ethylcellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose,hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate,cellulose acetate butyrate, cellulose acetate phthalate, carboxylethylcellulose, cellulose triacetate, cellulose sulphate sodium salt,poly(methyl methacrylate), poly(ethyl methacrylate),poly(butylmethacrylate), poly(isobutyl methacrylate),poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecylacrylate), polyethylene, polypropylene, poly(ethylene glycol),poly(ethylene oxide), poly(ethylene terephthalate), poly(vinylalcohols), polyvinyl acetate, poly vinyl chloride, polystyrene,polyvinylpyrrolidone, and polymers of lactic acid and glycolic acid,polyanhydrides, poly(ortho)esters, poly(butic acid), poly(valeric acid),and poly(lactide-cocaprolactone), and natural polymers such as alginateand other polysaccharides including dextran and cellulose, collagen,chemical derivatives thereof (substitutions, additions of chemicalgroups, for example, alkyl, alkylene, hydroxylations, oxidations, andother modifications routinely made by those skilled in the art), albuminand other hydrophilic proteins, zein and other prolamines andhydrophobic proteins, copolymers and mixtures thereof. In general, thesematerials degrade either by enzymatic hydrolysis or exposure to water invivo, by surface or bulk erosion.

Methods of Treatment

In one embodiment, the present invention provides a method of inhibitingan anti-tumor immune response mediated pathway target in a subjectcomprising the step of administering to the subject an effective amountof a PDE5 inhibitor, preferably as part of a composition additionallycomprising a pharmaceutically acceptable carrier. Preferably this methodis employed to treat a subject suffering from or susceptible to acondition selected from a disease or disease symptom (e.g., cancer,tumor, any disease or disorder delineated herein). Other embodimentsinclude any of the methods herein wherein the subject is identified asin need of the indicated treatment.

In another aspect, the method further includes administration of anadditional therapeutic agent. The additional therapeutic agent can be ananticancer agent, an anti-infective, or a PDE inhibitor.

Another aspect of the invention is a compound herein (e.g., PDEinhibitor, compound of any of the formulae herein) for use in thetreatment or prevention in a subject of a disease, disorder or symptomthereof delineated herein. Another aspect of the invention is the use ofa PDE5 inhibitor in the manufacture of a medicament for a disease ordisease symptom (e.g., cancer, tumor, any disease or disorder delineatedherein) in a subject. Preferably, the medicament is used for treatmentor prevention in a subject of a disease, disorder or symptom set forthabove.

Kits

The invention provides kits for the treatment or prevention of acondition associated with a disease or disease symptom (e.g., cancer,tumor, anti-tumor immune response, any disease or disorder delineatedherein). In one embodiment, the kit includes a pharmaceutical packcomprising an effective amount of a PDE5 inhibitor (e.g., a PDE5ainhibitor, such as sildenafil). Preferably, the compositions are presentin unit dosage form. In some embodiments, the kit comprises a sterilecontainer which contains a therapeutic or prophylactic composition; suchcontainers can be boxes, ampules, bottles, vials, tubes, bags, pouches,blister-packs, or other suitable container forms known in the art. Suchcontainers can be made of plastic, glass, laminated paper, metal foil,or other materials suitable for holding medicaments.

If desired compositions of the invention or combinations thereof areprovided together with instructions for administering them to a subjecthaving or at risk of developing a disease or disease symptom (e.g.,cancer, tumor, anti-tumor immune response, any disease or disorderdelineated herein). The instructions will generally include informationabout the use of the compounds for the treatment or prevention of adisease or disease symptom (e.g., cancer, tumor, anti-tumor immuneresponse, any disease or disorder delineated herein). In otherembodiments, the instructions include at least one of the following:description of the compound or combination of compounds; dosage scheduleand administration for treatment of a disease or disease symptom (e.g.,cancer, tumor, anti-tumor immune response, any disease or disorderdelineated herein); precautions; warnings; indications;counter-indications; overdosage information; adverse reactions; animalpharmacology; clinical studies; and/or references. The instructions maybe printed directly on the container (when present), or as a labelapplied to the container, or as a separate sheet, pamphlet, card, orfolder supplied in or with the container.

Here, it is demonstrated that PDE5 blockade represents a pharmacologictarget capable of down-regulating both Arg1 and NOS2 in tumorinfiltrating MSCs. This down-regulation abrogates tumor induced MSCsmediated immunosuppression and enhances tumor-specific immunity thatresults in measurable anti-tumor activity.

This is the first demonstration that blocking MSC-mediatedimmunosuppression via PDE5 inhibition imparts a measurable anti-tumorimmune effect. It is shown that PDE5-blockade increases intracellularcGMP resulting in degradation of NOS2 and suppression of NO production.Furthermore it also results in down-regulation of the IL4Rα-ARG-1pathway. Thus it targets both pathways critical to MSC function. Thesefindings establish a new role for PDE5 inhibition as a viable andeffective immunological adjunct in the treatment of various malignanciesadding to its therapeutic applications that already include thetreatment of erectile dysfunction, pulmonary hypertension (35, 36) andcardiac hypertrophy (18).

Although NO production from tumor-associated macrophages has bothtumor-promoting and tumoricidal properties, the ultimate effect of thesefree radicals is complex and likely dependent upon their localconcentration within the microenvironment. In fact, NO exerts itstumoricidal action through modulation of p53 expression (37).Interestingly, in a model in which human cancer cells were modified toexpress high levels of NO, cells containing wild type p53 demonstratedreduced tumor growth whereas cells with mutant p53 showed increasedproduction of vascular endothelial growth factor (VEGF),neovascularization and increased tumor growth (38). These studiesdemonstrate the duality of NO-mediated effects and its regulation by p53within a tumor setting. Unfortunately, most human tumors possess p53mutations (39) making them resistant to NO-mediated apoptosis.Furthermore, prolonged exposure to NO leads to the selection of a moreaggressive p53 mutant clone better able to escape the tumoricidal action(40). The clinical importance of NO-mediated antitumor efficacy isfurther limited by the fact that many human tumors such as melanoma,breast, stomach, ovarian and cervical cancers actually express NOS2. Infact, NO can promote cancer growth not only in the early stages of tumorprogression by facilitating DNA mutations (40) but also in the laterstages by increasing tumor angiogenesis (41) and immunosuppression (42).With regards to the immune escape mechanisms, it has been shown thatMSCs and/or tumor associated macrophages induce apoptosis or anergy inCD8⁺ and CD4⁺ T cells through a NOS2-dependent mechanisms (7, 20, 43).In fact, through inhibition of IL-2 signaling (20), NO productionanergizes Th1 T-cells. Alternatively, in a mixed Th1-Th2 environmentwhere arginase-induced pathways also mediate immunosuppression, MSCsproduce NO and super-oxide radicals to generate peroxynitrites thatinduce apoptosis of activated CD8⁺ T cells (12). With the growingunderstanding of the role of MSCs in tumor-induced immune dysfunction(6, 44, 45), targeted pharmacologic interventions have significantappeal in overcoming the suppressive mechanisms in immune-basedtherapeutic settings. It is recently shown that nitroaspirin couldabrogate the inhibitory activity of NO. It restored immuneresponsiveness in tumor-bearing hosts and enhanced the preventive andtherapeutic efficacy of antitumor vaccines (13). However, despite itsuse as a vaccine adjuvant, nitroaspirin demonstrated no anti-tumorefficacy when used alone.

PDE5 inhibition represents a novel immunopharmacologic target thatdown-modulates the expression of both Arg1 and NOS2 in MSCs.Interestingly, this approach more effectively reverses MSC-inducedimmune suppression than does nitroaspirin by exerting a significant invivo anti-tumor effect. The augmented anti-tumor effect can beattributed to the ability of PDE5 inhibition to target the varioussuppressive pathways by which MSCs inhibit T-cell function. To ourknowledge, this is the first demonstration that abrogation of MSCsuppressive mechanisms alone is sufficient to generate an antitumorimmune response. The measurable anti-tumor efficacy seen with PDE5inhibitors but not with NO inhibitors is likely due to the multi-targetinhibition exerted by these agents. Although results herein show thatPDE5 inhibitors affect both Arg-1 and NOS2 pathways in MSCs, it ispossible that additional as yet undefined pathways capable of furtherabrogating MSC-mediated immunosuppression may also be involved. Forexample, cGMP is also capable of reducing VEGF production within thehypoxic intra-tumoral environment (46).

One likely mechanism for the effect of PDE5-inhibitors on reducing NOproduction involves the impact of these inhibitors on mRNA stability.cGMP destabilizes NOS2 mRNA by reducing the ubiquitous mRNA bindingprotein, human-antigen-R (HuR) (47). HuR binds to AU-rich elements inthe 3′-untranslated region (UTR) thereby increasing the mRNA half-life(48). As such, destabilization of NOS2 mRNA via PDE5 inhibition wouldabrogate NO-mediated immunosuppression more effectively than wouldcompetitive inhibition of NO itself.

Since Arg1 mRNA does not possess AU rich elements nor has it beendescribed to be stabilized by HUR, other mechanism(s) are likelyinvolved in PDE5-mediated down-regulation of Arg1. One possibleexplanation is that high levels of cGMP induced by PDE5 blockade reducethe cytosolic Ca2+ concentration (49) leading to a reduction of thecalcium-dependent protein kinase C (PKC) activity (50) that in turnprevents up-regulation of IL4Rα (51). The link between IL4Rα and Arg-1in MSCs is supported by recent data demonstrating a direct correlationbetween ARG1 expression and IL4Rα expression. LysM^(Cre)IL4Rα^(−/flox)mice in which IL4Rα expression is knocked-out in neutrophils andmacrophages subsequently challenged with C26GM completely rejected thetumor when adoptively transferred with tumor-primed CD8+ T cells(Gallina et al. manuscript submitted). These data support our findingsby demonstrating that PDE5 blockade down-regulates IL4Rα expression ontumor-infiltrating MSCs (FIG. 1) and synergizes with the adoptivetransfer of tumor-primed CD8+ T cells (FIG. 4). This effect appears tospecifically target MSCs since IL4Rα expression on isolated CD11b⁺ cellsfrom tumor-bearing mice is significantly reduced when co-cultured in thepresence of sildenafil. Furthermore, the addition of IFN-γ, which invivo up-regulates IL4Rα expression through both autocrine and paracrine(presumably through activated T cells) mechanisms, is significantlyreduced in the presence of sildenafil (FIG. 7). Taken together thesedata underscore the critical role of the IL4Rα-ARG1 pathway in MSCs aswell as the use of PDE5 inhibitors as therapeutically effective drugs toovercome tumor-induced immunosuppression.

Effective adoptive cellular therapy requires T cells with thepredetermined antigen specificity to be present in sufficient numbers,traffic to the tumor site, and kill their target. Most solid tumors arecharacterized by a lymphocytic infiltration that is frequently unable tokill autologous tumor cells, indicating T cell anergy (52, 53), thepresence of regulatory T cells (Tregs) (54) or the existence of a non-Tcell immunosuppressive population. It was recently shown that humanprostate cancer anergic TILs can be reactivated in vitro through theinhibition of NOS2 and Arg1. These findings underscore importance ofMSC-mediated immunosuppression and identify putative targets ofimmunosuppressive pathways used by MSCs to improve immune-basedtherapeutic outcomes (11). These results are indicated by our in vivostudies whereby the sildenafil treatment led to an increase inintratumoral CD8⁺ T cell infiltration that inversely correlated withtumor size (FIG. 5b ), increased the percentage of activated T cells(FIG. 5c, and d ) and was the only condition in which adoptive celltransfer resulted in a measurable anti-tumor effect (FIG. 4).

The phenotype of human MSCs is still not well defined. However, there isevidence to suggest that a non-lymphoid CD34⁺ population plays a role inthe hypo-responsiveness of PBLs from head and neck cancer patients (55).A similar unresponsiveness is seen in PBLs from multiple myelomapatients (56). While the low proliferative capacity may be due tointrinsic T cell defects, a likely explanation for T cellunresponsiveness is the presence of a non-lymphoid suppressor accessorypopulation since PDE5-inhibition augments the proliferative index oflymphocytes from unfractionated peripheral mononuclear cell populationbut not of purified CD3⁺ cells (data not shown). Moreover, results fromour experiments suggest a prominent role of both Arginase and NOS2 in MMPBLs unresponsiveness. The ability of sildenafil to restoreCD3/CD28-stimulated proliferation of PBLs from both head and neck andmyeloma patients suggests that the mechanisms found in mice are alsopresent in humans.

Although different drugs such L-NMMA, Nor-NOHA, NO-aspirin, or VitaminD3 (57) have been used in vitro and in mouse models to alter the MSCssuppressive mechanisms, they have either not been extensively tested inhumans or found to be extremely toxic, as in the case with L-NMMA (58).Moreover the cytokines present in tumor microenvironment can be verydifferent among patient and tumor stage (59) and thus can promotedifferent suppressive pathways on MSCs. The use of safe and extensivelytested PDE5 inhibitors such as sildenafil, tadalafil, or vardenafil toovercome the different MSCs immune suppressive pathways is demonstratedby results described herein.

The following examples are provided to illustrate the invention, not tolimit it. Those skilled in the art will understand that the specificconstructions provided below may be changed in numerous ways, consistentwith the above described invention while retaining the criticalproperties of the compounds or combinations thereof.

EXAMPLES PDE5 Inhibition Down-Regulates NOS2 Expression inTumor-Associated MSCs

The primary property of MSCs is their ability to suppress an immuneresponse. While this phenotype is an essential defining feature,emerging data reveals varying degrees of immunosuppression of MSCsisolated from different organs. Tumor-associated MSCs express greaterlevels of NOS2 and Arg-1 than do splenic MSCs and, thus, result ingreater immune suppression (data not shown). Since cGMP analogues canreduce NO generation in monocytes (15), this investigation sought todetermine whether NO production in tumor-associated MSCs could bereduced with the in vivo treatment of the PDE5 inhibitor, sildenafil (20mg/kgday). BALB/c mice were challenged subcutaneously with the coloncarcinoma, CT26WT. Half the mice were then treated with sildenafil. Themice were sacrificed 15 days later and intratumoral MSCs were purifiedfrom the single cell suspensions. MSCs derived from the sildenafil groupshowed higher intracellular cGMP levels than the control group asassessed by a competitive enzyme immune assay (FIG. 1a ). No significantdifferences were seen in the non-MSC population (data not shown).Interestingly, higher cGMP concentrations correlated withdown-regulation of NOS2 (FIG. 1b ) and lower NO production (FIG. 1c ) bythe intratumoral MSCs. Surprisingly, sildenafil treatment alsodown-regulated Arg-1 (FIG. 1b,c ) the other gene involved in MSCmediated immunosuppression. Arg-1 expression is mainly regulated by theSTAT6-IL4Rα pathway (19) and data by Gallina et al. recently correlatedIL4Rα expression on CD11b/GR1 with the immunosuppressive phenotype(manuscript submitted). IL4Rα expression via flow cytometry on purifiedtumor infiltrating MSCs from untreated or sildenafil treated mice wasanalyzed. IL4Rα is up-regulated in tumor infiltrating MSCs as comparedto splenic MSCs from tumor free mice. In contrast, sildenafil treatmentsignificantly reduced IL4Rα expression on the intratumoral MSCs and thiscorrelated directly with the decrease in Arg-1 expression and activity.In addition to confirming the role of IL4Rα in MSC mediatedimmunosuppression, its expression in splenic MSCs cultured alone or inpresence of sildenafil (FIG. 7) was analyzed. Sildenafil not onlydown-regulates IL4Rα in cultured MSCs, but also prevents IFN-γ mediatedIL4Rα up-regulation—a necessary process for MSC-mediated immunesuppression (Gallina et al. submitted). These findings reveal a novelmechanism by which MSCs suppressive pathways can be pharmacologicallyregulated both in vivo and in vitro: PDE5 inhibition up-regulatesintracellular cGMP and decreases NOS2 and Arg1 protein levels—themediators of MSC suppression.

In Vitro PDE5 Inhibition Abrogates MSC Immunosuppression.

Freshly isolated MSCs from tumor-bearing mice suppress the in vitroproliferation of activated lymphocytes. The exact mechanisms ofsuppression appear to be strain specific: in the Th-1 prone strain,C57Bl/6, it is mediated by NOS2 through NO production (via the NOS2catalytic domain) (20). Whereas, in the mixed Th-1/Th2 Balb/c strain,suppression is mediated either by peroxynitrite formation (via Arg1 andNOS2 co-expression) (10) or by L-arginine depletion secondary to Arg1over-expression (21). By reducing both Arg1 and NOS2 expression, PDE5inhibition affects all these suppressive pathways resulting in reducedMSC-mediated immunosuppression and enhances antigen-specific T cellresponsiveness. Tumor-derived CD11b⁺ MSCs were isolated from BALB/c micebearing the colon carcinoma, C26-GM. We utilized the irradiated, C26cell line retrovirally transduced to produce GM-CSF since this cytokinehas been shown to recruit MSCs more rapidly than the unmodified CT26cell line (10, 22, 23). We tested MSC suppressive activity by admixingMSCs with CFSE-labeled hemagluttinin (HA)-specific CD8⁺ (clone 4) orCD4⁺ (6.5) T cells pulsed with their relevant peptide in the presence orabsence of sildenafil (FIGS. 2a and b ). Whereas the addition oftumor-derived MSCs significantly impaired antigen specific T cellproliferation as demonstrated by the low percentage of CFSE^(lo)clonotypic T cells, sildenafil almost completely restored both CD4⁺ andCD8⁺ responsiveness of antigen-specific T cells. The absence of asildenafil-mediated enhancement in T cell function in the groups lackingCD11b cells underscores the targeted role of sildenafil on the MSCpopulation. In an effort to understand the in vivo mechanisms mediatingthis effect, we utilized C57Bl/6 mice for several reasons: 1) in thisstrain, inhibition of NOS2 is sufficient to revert MSCs mediatedimmunosuppression (7); and 2) NOS2^(−/−) mice are available thusenabling us to examine the effect of PDE5 blockade in the NO-mediatedpathway of immunosuppression. CD11b⁺ MSCs were isolated from eitherB16GM melanoma-bearing C57BL/6-NOS2^(+/+) or B16GM melanoma-bearingC57BL/6-NOS2^(−/−) mice. A suppression assay was performed bystimulating OVA-specific CD4⁺ T-cells with the relevant peptide in thepresence or absence of MSCs derived from either NOS2^(+/+) or NOS2^(−/−)mice (FIG. 2c ). While the addition of C57Bl/6-NOS2^(+/+)-derived MSCsinduced considerable T cell suppression, no suppression was observedwith MSCs from NOS2^(−/−) mice. Furthermore, while PDE5 inhibitionreversed MSC suppression in NOS2^(+/+) mice, the addition of sildenafilto the NOS2^(−/−)-derived MSC suppression assay did not augment T cellresponsiveness. Taken together, these results confirm the role of NOS2in MSC-mediated T cell suppression (FIG. 2c ) and demonstrate that PDE5inhibition can revert two different suppressive pathways (Arg1 and NOS2)by which MSCs impair immune responsiveness.

In Vivo PDE5 Inhibition Delays Tumor Outgrowth by an Immune MediatedMechanism.

Having recently shown that the in vivo inhibition of the MSC suppressivepathways by nitroaspirin was ineffective as a single agent but augmentedthe anti-tumor efficacy of vaccines on established tumors (13), it wassought to determine whether PDE5 inhibition alone, by affecting bothArg-1 and NOS2 suppressive activity, could impart a measurableanti-tumor effect. Mice were challenged either with CT26-WT or with themore aggressive tumor, C26GM and then treated with PDE5 inhibitors. Asshown in FIG. 3a and FIG. 8, sildenafil or tadalafil treatment alonesignificantly delayed tumor outgrowth through an immune mediatedmechanism as evidenced by the lack of anti-tumor efficacy in the immunedeficient Rag^(−/−) mice (FIG. 3b ). However, the combination of PDEinhibition with the MSC-depleting anti-GR-1⁺ antibody conferred nosynergistic effect (FIG. 3c ). Taken together, these data confirm thatMSC-mediated immunosuppressive pathways function via NOS2-Arg1 enzymaticactivity produced by GR-1⁺ cells and demonstrate the ability of PDE5inhibition to abrogate their activity in vivo. To the best of ourknowledge, this is the first demonstration that a direct anti-tumoreffect can be obtained through the pharmacologic inhibition oftumor-induced immunosuppressive pathways.

PDE5 Inhibition Enhances Anti-Tumor Efficacy of Adoptive Immunotherapy.

Adoptive immunotherapy of tumor-specific T cells offers much promise asa therapeutic modality. Considerable progress has been made indeveloping strategies to isolate, expand and activate tumor specificcells in vitro. In the appropriate environment, these lymphocytes canmediate significant tumor destruction. However, the mere presence oftumor reactive T cells in the peripheral circulation is not sufficientto induce tumor rejection (24). T cells must also traffic to the tumorsite and overcome the intrinsic immunosuppressive barriers toeffectively kill in situ. To determine whether sildenafil inhibition ofthe MSC suppressive pathways could improve the efficacy of adoptiveimmunotherapy, 20×10⁶ C26GM-primed splenocytes were transferred intoC26GM bearing animals. Following adoptive transfer, the mice were eithertreated with sildenafil or left untreated. As shown in FIG. 4, adoptivetransfer alone demonstrated no anti-tumor efficacy whereas PDE5inhibition showed a statistically significant reduction in tumoroutgrowth. However, coupling adoptive immunotherapy with PDE5 inhibitionyielded the greatest anti-tumor efficacy. These data suggest thatdisruption of the MSC-mediated, immunosuppressive microenvironment iscritical to augment the therapeutic efficacy of adoptive immunotherapyin cancer-bearing hosts.

PDE5 Inhibition Increases the Number of Tumor Infiltrating CD8⁺ Cells.

Tumor specific T-cells must be present in sufficient numbers and capableof trafficking to their targets to exert a measurable antitumor effect.In fact, a direct correlation exists between the number of infiltratinglymphocytes and a favorable clinical outcome in patients with metastaticovarian cancer (25). Moreover, the presence and the functionality oftumor infiltrating lymphocytes (TILs) correlates with a favorableprognosis in various human malignancies (25-29). Since PDE5 inhibitionaugments anti-tumor immunity, whether sildenafil treatment altered thenumber and/or the activation state of TILs was considered.Hematoxylin-eosin staining revealed a greater intratumoral cellularinfiltration in the sildenafil-treated mice compared to the untreatedcontrols (FIG. 5a ). To better evaluate these differences, tumor-bearingmice either received tumor-primed T cells or no T cells followed bysildenafil treatment or no additional therapy. The tumor was thenexcised and single cell suspensions were obtained. The T cellinfiltration was analyzed by flow cytometry for CD4⁺ and CD8⁺ T cells.This approach enabled us to accurately examine the entire tumor mass andreliably quantify the infiltrating lymphocytic population. Sildenafiltreatment resulted in a greater CD8⁺ tumor infiltration. Interestingly,no increase in CD4⁺ T cells was observed with PDE5 blockade (FIG. 5binsert). Moreover, sildenafil significantly activated thetumor-infiltrating CD8⁺ T cells as revealed by up-regulation of bothCD69 and CD25 activation markers (FIG. 5c ). There were no differencesin activation markers between the sildenafil-treated group andsildenafil plus adoptive cell therapy (ACT). These data indicate thatthe anti-tumor efficacy in the sildenafil+ACT group (FIG. 4 and FIG. 5b) is primarily attributable to sildenafil's ability to abrogate theimmunosuppressive mechanisms within the tumor microenvironment.

Interestingly, in the advanced tumor setting, the percentage of tumorinfiltrating CD8⁺ T cells negatively correlated (Spearman bivariatecorrelation P<0.001) with tumor size which supports the concept ofsildenafil's ability to create a more permissive immune environment(FIG. 5b ). The maximal therapeutic effect was seen in the ACT groupwhere a larger CD8⁺ T cell infiltrate was present in the tumor.Moreover, tetramer staining suggested that the tumor-infiltrating CD8⁺more effectively recognized the tumor associated antigen in thesildenafil-treated group. In fact, 9.08%±0.905 of tumor-infiltratingCD8⁺ cells were specific for AH1, one of the major C26GM-associatedantigens, whereas only 1.19±1.180 were tetramer positive in theuntreated group (n=3).

IL-2 is required for the activation of naïve T cells and generates alymphocyte population with heightened recall responses. Furthermore,IL-2 production is associated with the persistence of tumor specificCD8⁺ lymphocytes within the tumor microenvironment and systemicadministration of low doses of IL-2 improves the persistence andantitumor efficacy of transferred T cells (30). It was previously shownthat NO can alter IL-2 production in activated lymphocytes. To examinewhether the immunomodulatory effect of PDE5 inhibition affected T cellactivation within the tumor microenvironment, IL-2 production by TIL wasexamined. To accomplish this, a transgenic mouse model of greenfluorescent protein (GFP) under an IL-2 promoter (BALB/c-IL-2p/GFP) (31)was utilized. In this model, T cell stimulation results in activation ofthe IL-2 and expression of the reporter transgene GFP, easily detectableby flow cytometry. C26GM-primed BALB/c-IL-2p/GFP splenocytes wereadoptively transferred to tumor-bearing recipients that were either leftuntreated or treated with sildenafil for 9 days. Single cell suspensionsof the tumor-infiltrating CD8⁺ were analyzed by FACS for GFP expression.Adoptively transferred, vaccine-primed T cells were activated in thetumor microenvironment only with PDE5 inhibition whereas, in itsabsence, they were unable to release IL-2, and hence were bona fideanergic T cells (FIG. 5d ). To further prove that these effects weredependent on CD8⁺ T cells, mice were challenged with C26GM andeither: 1) left untreated; 2) given sildenafil; 3) an anti-CD8 depletingantibody; or 4) both. Sildenafil treatment again demonstrated astatistically significant reduction in tumor outgrowth, an effectcompletely abolished by CD8⁺ depletion (FIG. 5e ). These experimentsindicate that the in vivo MSC suppressive pathways limit T cellinfiltration, activation and anti-tumor efficacy. Abrogating thesesuppressive mechanisms via PDE5 inhibition enhances the tumor specific Tcell response and generates a measurable anti-tumor response.

T Cell Proliferation is Restored by PDE5 Inhibition in Multiple Myelomaand Head and Neck Cancer Patients.

Head and neck cancers express high levels of GM-CSF responsible for theintratumoral infiltration by CD34⁺ MSC suspected of playing a major rolein the immune suppression observed in these patients (32). In fact, ithas been shown, that peripheral blood lymphocytes (PBLs) from thesepatients are functionally impaired in that they fail to be activated andproliferate poorly upon stimulation (33). This anergic state is in largepart due to the Arg1 and/or NOS-dependent suppressive activity of MSCs(21, 34). Similar results were also seen in prostate cancer (11) and inmultiple myeloma patients (Noonan, unpublished data). It was sought todetermine whether one could restore T cell proliferation of PBLsisolated from head and neck and multiple myeloma patients and stimulatedwith anti-CD3 and anti-CD28 antibody-coated beads, in the presence orabsence of sildenafil. While the addition of sildenafil to the culturehad no effect on PBLs from healthy donors, PDE5 inhibition significantlyrestored CD4⁺ and CD8⁺ proliferation in all the examined patients (FIG.6). Interestingly, the addition of sildenafil to isolated CD3⁺ T cellswas unable to increase T cell proliferation (data not shown). Takentogether, these human data confirm that the PDE5 inhibition augmentsimmune responsiveness through its effect on an accessory, non-T cellpopulation. Moreover, these data suggest that the same immunosuppressivemechanisms found in mice are conserved in human malignancies and thatPDE5 can be a useful therapeutic target to improve anti-tumorimmunotherapy.

Cell Lines:

CT26 and C26-GM are BALB/c colon carcinoma cells lines previouslydescribed (10). B16-GM is a C57Bl/6 melanoma cell line previouslydescribed (60). Cells were grown in DMEM (Invitrogen Carlsbad, Calif.)or in RPMI medium 1640 (Invitrogen) supplemented with 2 mML-glutamine/10 mM Hepes/20 μM 2-mercaptoethanol/150 units/mlstreptomycin/200 units/ml penicillin/10% heat-inactivated FBS (Harlan,Indianapolis Ind.).

Drugs and Cytokines:

Sildenafil (Pfizer, New York N.Y.) was dissolved in the drinking water(20 mg/kg/24 h), given intraperitoneally (ip) daily where indicated (20mg/kg/24 h) or added to the cell cultures at a final concentration of 50mg/ml. Tadalafil (Lilly ICOS. Bothel Wash.) was given ip at aconcentration of 2 mg/kg/24 h. IFN-γ (25 ng/ml)(Peprotech, Rocky Hill,N.J.) was added where indicated.

Mice and In Vivo Experiments:

4-6 weeks old Balb/c mice were purchased from Harlan. C57Bl/6-NOS2^(−/−)mice (strain B6; 129P2-Nos2^(tm1Lau)) or the control mice (strainB6129PF2/J 100903) were purchased by Jackson Laboratories (Bar Harbor,Me.). Rag−/− were bred in the Johns Hopkins animal facility.Balb/c-pIL2-GFP mice were a kind gift of CT. Weaver (University ofAlabama)(31). pCL4-TCR mice are transgenic for an influenza virusHA512-520 peptide-specific, H-2Kd-restricted TCR composed of V10 andVβ8.2 chains were described before (23). All experiments involving theuse of mice were in accordance with protocols approved by the AnimalCare and Use Committee of the Johns Hopkins University School ofMedicine. Tumor measurements were performed in a blind fashion with acaliper by measuring the two main diameters and tumor size is expressedas their product. Mice were euthanized for ethical reasons when tumorsize was greater than 150 mm². GR1 depletion was performed by ipinjection of 100 μg of anti-GR-1 depleting antibody (clone RB6.8C5-18).This clone was obtained by sub-cloning the originally described RB6.8C5to maximize the antibody production. CD8 depletion was performed by ipinjection of 200 μg of anti-CD8 depleting antibody (clone 2.43) on days0, 2, 4, 6. The antibodies were produced in vitro in protein-free medium(Invitrogen), purified by G-protein affinity chromatography, andquantified utilizing a Rat IgG2b ELISA (Bethyl Inc Montgomery, Tex.).Adoptive cell transfer (ACT): Donor mice were tumor primed bysubcutaneous (sc) injection of 10⁶ γ-irradiated C26GM into four limbsone week prior to adoptive T cell transfer. For the adoptive transferexperiments, lymph nodes and spleens were harvested and mechanicallydisrupted to obtain single cell suspensions. The cells then underwentRBC lysis with ACK Lysing buffer (Biosource, Camarillo Calif.) and20×10⁶ cells were injected i.v. into each recipient.

Collagenase Treatment of Tumors:

Single cell suspensions were obtained from the tumors by collagenasetreatment. Briefly, tumors were surgically removed, and incubated 30 min@ 37 C.° with a solution of collagenase (10 mg/ml Collagenase, 0.1 mMMgCl2 0.1 mM CaCl₂) coupled with mechanical disruption. The reaction wasstopped with 10 vol. of medium containing 10% fetal calf serum. Thecells were washed, red blood cells lysed, and the cell suspensions werepassed through a cell strainer. For hematoxylin-eosin staining, wholetumors were washed twice with PBS and then incubated for 5 days with 10volumes of 10% neutral buffered formalin.

Flow Cytometry:

Single cells suspensions from spleens or tumors were stained withPhycoerythrin (PE)-conjugated anti-mouse CD8 (CD8-PE) (BD-Pharmingen SanJose, Calif.), Allophycocyanin (APC)-conjugated anti-mouse CD4 (CD4-APC)(BD-Pharmingen) or with APC conjugated anti-mouse CD11b (BD) andPE-conjugated anti-mouse Gr-1 (CD8-PE). IL4Rα expression was evaluatedon purified CD11b⁺ cells by the use of an anti-mouse CD124 PE-conjugated(BD-Pharmingen). Isotyped matched antibodies were used as controls andliving cells were gate based on 7AAD, annexinV staining. A total of100,000 events were collected for each sample on a FACScalibur (BD) flowcytometer, and the data were analyzed using FCS express v2.0 (De-novosoftware).

Cell Purification:

CD11b⁺ purification was performed with the Mouse CD11b MicroBeads(Miltenyi Biotec, Bergish-Gladbach, Germany), positive and negativefractions were sorted with the LS columns following the manufacturer'sinstructions. Suppressive assay: Purified splenic CD11b⁺ cells (2×10⁵)were added to CFSE-labeled splenocytes (10⁶) derived from C14 transgenicmice stimulated for 3 days with Class I HA₅₁₂₋₅₂₀ peptide (IYSTVASSL) in96 flat bottomed plates. Sildenafil was added where indicated.Proliferation assay: PBLs were obtained from head and neck or multiplemyeloma cancer patients having obtained informed consent using anInstitutional Review Board-approved protocol. T-cell stimulation wasperformed by adding anti-CD3/CD28 antibody coated Dynal beads toficolled PBLs suspended in serum free media at a 3:1 bead to T-cellratio. The cells were cultured for 5 days in a 96 round-bottom wellplate at 37 C 5% CO2. The cells were then labeled with CD4 and CD8antibodies and analyzed by flow cytometry. Sildenafil was added whereindicated. Results are reported as fold change (number of activatedcells/number of unactivated cells).

cGMP was measured on purified CD11b⁺ cells using the “Cyclic GMP EIA”Kit (Cayman Chemical Ann Arbor, Mich.). Data analysis was performed withthe workbooks available athttp://www.caymanchem.com/neptune/servlet/neptune/template/analysis%2CEIA.vm/a/z.Data are expressed as mean+/−SE of quadruplicate wells.

Western Blot:

Cells (10⁶) were purified and washed twice with PBS, and the pellet wasresuspended in PBS with 0.2% Triton X-100 and 2 mM EDTA, and incubatedfor 10 min at room temperature. Cell lysates were centrifuged at 14,000rpm for 1 min, and 1 vol of Laemmli's sample buffer (4% SDS, 20%glycerol, 10% 2-ME, 4 mg/100 ml bromophenol blue, and 125 mM Tris-HCl;pH 6.8) was added to the supernatant. After incubation at 95° C. for 10min, lysates were subjected to SDS-PAGE, and proteins were transferredovernight to PVDF membranes. The membranes were saturated at RT inPBS/0.05% Tween 20 containing 2% nonfat milk (Sigma-Aldrich) for 1 hr.The membranes were then incubated overnight at room temperature withrabbit polyclonal anti-NOS2 (Santa Cruz Biotechnology Santa Cruz,Calif.), mouse anti-Arg1 (a kind gift from Augusto C. Ochoa, LouisianaState University, New Orleans, La.) or polyclonal rabbit anti-actinantibody (Sigma-Aldrich), washed twice with PBS/0.05% Tween 20, andincubated with the either mouse anti-mouse IGG or donkey anti rabbit IGGHRP-linked secondary antibody (Amersham Biosciences, Little Chalfont,U.K.) for 1 h at room temperature. Proteins were detected using theSupersignal West picochemiluminescent substrate kit (Pierce, Rockford,Ill.) according to the manufacturer's instructions.

NO Measurement:

NO was measured using a nitrate/nitrite assay kit (Cayman) according tothe manufacturer's instructions. Results were normalized to 10⁶ cells.Data are from triplicate wells.

Arginase Assay:

CD11b⁺ cells were magnetically purified from the surgically removedtumor, washed twice in PBS-BSA 1%, counted and lysed with Triton X100,0.1%. The arginase assay was performed as previously described (10).

Statistical Analysis:

Bivariate Pearson and ANOVA analysis were performed using SPSS v7.0 Allexperiments were repeated at least twice and all the P values weretwo-sided (T test) or one-sided (Anova).

All references cited herein, whether in print, electronic, computerreadable storage media or other foot′, are expressly incorporated byreference in their entirety, including but not limited to, abstracts,articles, journals, publications, texts, treatises, technical datasheets, internet web sites, databases, patents, patent applications, andpatent publications.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable herein includes that embodiment as any single embodimentor in combination with any other embodiments or portions thereof.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

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1. A method of treating or preventing cancer in a subject comprisingadministration to the subject of a PDE-5 inhibitor compound.
 2. Themethod of claim 1, further comprising administration to the subject ofan additional anticancer agent.
 3. The method of claim 2, wherein theadditional anticancer agent is an immunotherapeutic agent.
 4. The methodof claim 3, wherein the additional anticancer agent is a vaccine.
 5. Themethod of claim 1, wherein the subject is identified as in need oftreatment of cancer with a PDE-5 inhibitor compound.
 6. The method ofclaim 1, wherein the PDE-5 inhibitor compound is selected fromsildenafil, vardenafil or tadalafil.
 7. A method of modulating myeloidsuppressor cells (MSCs) immune suppression in a subject comprisingadministration to the subject of a PDE-5 inhibitor compound.
 8. Themethod of claim 7, wherein the modulating is down-modulation. 9-16.(canceled)
 17. A method of treating or preventing disease in a subjectcomprising administration to the subject of a PDE-5 inhibitor compound.18. The method of claim 17, wherein the disease is a disease mediated bymyeloid suppressor cells (MSCs).
 19. The method of claim 18, wherein thedisease is cancer, chronic infection, chronic inflammation,hematopoietic reconstitution following chemotherapy.
 20. The method ofclaim 1, further comprising the step of assessing MSC levels in thesubject.
 21. The method of claim 1, further comprising the step ofassessing MSC levels in the subject before and after administration. 22.The method of claim 20 or 21, wherein the assessment is by surfacemarker expression.
 23. The method of claim 20 or 21, wherein theassessment is by MSC number.
 24. The method of claim 20 or 21, whereinthe assessment is by measure of immunosuppression function.
 25. Themethod of claim 1, wherein the cancer is multiple myeloma, lymphomas,melanoma, breast, stomach, head and neck, ovarian, colon, prostate,lung, high grade gliomas, or cervical cancer. 26-32. (canceled)